Life Sciences Applications

CHAPT ER

19 Life Sciences Applications Jianhua Wang and Xuwei Chen

Contents

1. Introduction 2. Deoxyribonucleic Acid (DNA) Assays 2.1 DNA separation and purification from biological matrices 2.2 DNA quantification 2.3 DNA separation and amplification 3. Assays of Proteins, Peptides and Amino Acids 3.1 Quantitative assay protocols 3.2 Activity measurements 3.3 Separation of biomolecules 3.4 Protein immobilization 4. Immunoassays 5. Enzymatic Assays 5.1 Heterogeneous enzymatic assays based on microreactors 5.2 Homogeneous enzymatic assays 6. Cellular Analysis 7. Perspectives Abbreviations References

559 560 560 562 565 566 566 569 569 575 575 581 581 583 585 586 587 588

1. INTRODUCTION Flow injection analysis (FIA), based on automatic injection of a series of samples/ reagents into a continuous carrier stream, has been developed as a powerful technique for fluidic manipulations prior to detection. Unlike conventional solution handling procedures based on equilibrium of the reaction systems, FIA obtains the analytical data from dynamic processes taking place in the flow manifolds, thus the analysis time is greatly reduced and at the same time, better precision, higher sample throughput and reduction in sample consumption are achieved. It has provided a versatile and inexpensive methodology for automation of analytical procedures and the advantages mentioned herein could readily explain the explosive growth of publications concerning FIA [1]. Comprehensive Analytical Chemistry, Volume 54 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00619-3

r 2008 Elsevier B.V. All rights reserved.

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In 1990, the conception of sequential injection analysis (SIA) was conceived in order to reduce the inconvenience that hindered the utilization of FIA as a routine analytical tool. Relatively large consumption of reagents in the FIA mode is avoided since only the required amounts of reagents are aspirated and carrier is not pumped continuously in the SIA protocol. At the same time the SIA methodology offers great advantages since the systems can be easily adapted to various analytical protocols by merely adjusting and controlling the flow parameters through a computer without changing the physical configuration. The bead injection analysis (BIA) scheme has been recently developed as a more flexible technique, in which manipulation of beads takes place in flow-based manifolds. The BIA technique not only offers high precision for beads delivery but also avoids carryover in repetitive sample handling. The automated transportation of solid materials within the flow system facilitates well their renewal whenever necessary and provides a high degree of repeatability when metering, packing and perfusion of beads with samples and reagents. In addition, some beads, such as Sephadexs and Sepharoses can be detected directly in situ by UV–VIS and fluorescence spectrometry, which allows real-time monitoring of binding and elution of analytes. The flow-based techniques outlined above have proved to be powerful fluidic manipulating approaches, which have been discussed extensively in a number of articles [2,3]. They also offer an elegant and versatile interface for various detection techniques [4,5]. In the automatic mode, the FIA/SIA systems play very important roles by replacing labor-intensive manual procedures. In addition, they are especially suitable for reliable long-term operations. Features such as low sample and regents consumption, reduced analysis time, favorable reproducibility and repeatability and minimal sample contamination in a closed processing system, obviously make the FIA and SIA techniques among the most suitable approaches in life sciences analysis. In the last decade, the three generations of flow analysis techniques (i.e., FIA, SIA and lab-on-valve (LOV)), have been widely employed in life sciences analysis, including immunoassays and assays of various target macrobiomolecules (e.g., nucleic acids, protein species as well as amino acids).

2. DEOXYRIBONUCLEIC ACID (DNA) ASSAYS 2.1 DNA separation and purification from biological matrices The extraction and purification of DNAs from a variety of complex sample matrices (real-world biological samples) is a critical process, which should provide pure solutions of the DNAs of interest for further biological investigations. These solutions should be free of interferences arising from coexisting soluble constituents. These biological investigations may include processes such as polymerase chain reactions (PCR) and DNA hybridization. At this point, solidphase extraction (SPE) based on the affinity adsorption and separation of DNAs has been proven to be the most straightforward, robust, simple and efficient

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purification methodology, which can readily be performed in an appropriate FIA/SIA manifold. An FIA procedure including ion-exchange purification of nucleic acids in a miniaturized expanded-bed column has been developed by Nandakumar et al. [6] for on-line monitoring of the concentration of plasmid DNA during the cultivation of E. coli. In this case, the sorbent material was treated as a stationary component, which was used repetitively for sorption-dissolution of DNA and was renewed only after numerous analytical runs. The introduction of a renewable surface technique in FIA/SIA systems involving SPE with regeneration of the sorbent material after each analysis was reported [7,8]. This approach facilitated efficiently the avoidance of a problem frequently encountered in conventional SPE operations, which stemmed from the contamination or deactivation of the sorbent surface after processing a large number of samples. In some cases the processing of numerous samples can also lead to the loss of functional groups or active sites. Chandler and coworkers developed an SIA system with a renewable separation column (SIA–RSC) for automating the purification of total DNA from complex matrices [9–11]. A rotating rod, as depicted in Figure 1, was employed for the renewal of the solid phase, which was aimed at improving the reproducibility. According to the authors, this micro-column equipped with a rotating rod was capable to handle particulate materials without any clogging thus providing reproducible performance for a few months. This system was also adopted for studying

Figure 1 Schematic diagram of a rotating rod renewable filter (A). With the beveled rod in the trap position, beads with diameter larger than the leaky tolerance are collected in the microcolumn as the fluid flows continuously through the outlet port. To flush the beads to waste, the beveled rod is rotated at 1801 (B). Reprinted from [1]. Copyright (2003), with permission from Elsevier B.V.

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solid-phase nucleic acid binding chemistry [12]. Rapid evaluation of variables including solution compositions and temperatures of hybridization and elution was achieved. As the third generation of flow analysis techniques, LOV systems with their versatile channel design facilitate fluidic manipulation at the level of 0.1B100 mL, thus allowing surface renewal in a more flexible and precise manner. An LOV system, integrating a renewable micro-column, as illustrated in Figure 2, has offered significant advantages for DNA separation and purification in terms of improved sampling frequency, long-term stability and better precision over chipbased and capillary-based separation/purification systems utilizing the same principles [13]. In addition, the employment of BIA in an LOV system further facilitated real-time renewal of the micro-column thus leading to a significant improvement in the long-term reliability and robustness of operation. With a fluidic manipulation at the meso-fluidic level, the LOV-based DNA purification system also presents itself as an excellent sample pretreatment front end for microfluidic analysis systems such as micro-chip PCR and/or micro-chip electrophoresis. These systems provide a promising platform for the integration of DNA purification, PCR amplification and micro-chip electrophoresis into a compact system [13,14].

2.2 DNA quantification The accurate quantification of trace amounts of nucleic acid is a critical step in a wide variety of biological and diagnostic applications such as genetic diagnosis

Figure 2 Schematic diagram of the LOV meso-fluidic system with integrated demountable fluorescence flow cell, employed for DNA separation and purification (EtBr, ethidium bromide; LIF, laser-induced fluorescence; P1, P2, peristaltic pumps; SP, syringe pump; V1, three-way valve). Reprinted from [13]. Copyright (2003), with permission from Springer Science and Business Media.

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and forensic analysis. Considering the limited amount of source material for biological samples, rapid analysis protocols and the avoidance of cross contamination are crucially required. Obviously these requirements cannot be fulfilled by conventional manual procedures. FIA/SIA techniques show clear advantages for the quantification of DNA at trace levels because of the possibility for automatic fluidic manipulation and minimized cross contamination. A variety of detection techniques have been adopted for DNA quantification in flow-based analysis systems. These include spectrophotometry [15], spectrofluoromerty [16–21], chemiluminescence [22–26] and amperometry [27,28] as summarized in Table 1. Due to the controllable and highly reproducible dispersion in FIA systems, a higher sampling frequency is usually obtained and the reproducibility is improved, but the consumption of reagents is somewhat relatively high when a continuous flow of reagent and carrier takes place. This results in high analysis expenditures and the production of large amounts of toxic waste. In order to overcome these problems, some promising techniques have been developed such as the renewable drops technique [18]. In this technique the DNA and reagents solutions are delivered into a silica capillary tube by a peristaltic pump to form drops at the tip of the capillary tube where fluorescence measurements are conducted by employing optical fibers. This windowless detection system employing renewable drops provides a fresh reaction surface for each sample. This is of particular value for solving problems arising from irreversible reactions. At the same time lower reagents consumption is easily achieved. The versatility of in-valve spectrometric detection and precise fluidic manipulation make the LOV system a suitable alternative for reducing reagents and sample consumption. This has been well demonstrated in DNA quantification studies. A novel procedure with spectophotometric or fluorometric detection carried out in a meso-fluidic LOV system has been developed recently [15,21]. In the spectrophotometric detection mode, only 10 mL of reagent (crystal violet solution) and 5.0 mL of sample solution were required for each analysis; while in the fluorometric mode with laser-induced detection, the sampling volume was further downscaled to nano-liter levels, i.e., 600 nL, while at the same time much higher sensitivity (10-fold improvement) was achieved. Recently, an LOV procedure for the specific detection of single-stranded nucleic acid sequences via sandwich hybridization was proposed by Edward and Baeumner [19]. UV and fluorescence detection has been exploited for monitoring the on-bead oligonucleotide hybridization as well as for the quantitative analysis of DNA strands with a linear dynamic range of 1–1,000 pmol. Song et al. [29] proposed an FIA manifold coupled to a ultra-sensitive surface plasma resonance (SPR) spectrometer for the detection of sequence-specific ultratrace levels of oligodeoxynucleotides and polydeoxynucleotides. A miniaturized flow cell (with a capacity of 4 mL) was constructed as an interface between the detector and the flow system. A detection limit at 54 fmol L1 was obtained, which implies a significant improvement in the detectable concentration levels by 2–3 orders of magnitude as compared to some of the other SPR methodologies.

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Table 1

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Applications of flow-based techniques in quantitative assays of DNA

Detection method and comments

Calibration range

Detection limit

Sampling frequency

References

Absorbance, decoloration of DNA on crystal violet, 591 nm Fluorescence enhancement of berberin after reaction with DNA, lex/em ¼ 362/ 531 nm Fluorescence enhancement of Hoechst 33258 after reaction with DNA Fluorescence quenching of TMB-da after adding DNA, lex/em ¼ 278/ 403 nm Fluorescence enhancement of fluorescein after reaction with DNA, lex/em ¼ 480/ 520 nm Fluorescence enhancement of Ru(bpy)2PIP(II) after reaction with DNA, lex/em ¼ 460/ 590 nm Fluorescence enhancement of ethidium bromate after reaction with DNA, lex/ em ¼ 473/610 nm Chemiluminescence of the Rhodamine BCe(IV)-DNA system

0.2–6.0 mg mL1

0.07 mg mL1

30 h1

[15]

0–12 mg mL1

7.3 ng mL1

60 h1

[16]

–

0.01 mg

–

[17]

0.03–8.4 mg mL1

10 ng mL1

–

[18]

1–1,000 pmol

1 pmol

–

[19]

0–4 mg L1

3.7 mg L1

60 h1

[20]

0.03–3.0 mg mL1

0.009 mg mL1

60 h1

[21]

1.0  10–8– 0.1 mg mL1 2.1  10–6– 0.21 mg mL1 2.6  10–5– 0.26 mg mL1

8.3  10–9 mg mL1

–

[22]

3.5  10–7 mg mL1

[23]

6.5  106 mg mL1

[24]

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Table 1 (Continued )

a

Detection method and comments

Calibration range

Detection limit

Sampling frequency

References

Chemiluminescence of the Ce(IV)-Na2SO3Tb(III)-fluoquinolone antibiotic system Chemiluminescence of the 9,10anthraquinone-2,6disulphonic acidDNA system Amperometric detection of the oxidation of bound guanine moiety Electrochemical detection of the adsorption/ desorption of DNA on polypyrrole (PPy)coated electrodes

0.04–10 mg mL1

7.8 mg mL1

–

[25]

0.04–5.5 mg mL1

17 ng

45 h1

[26]

–

460 pg

–

[27]

1–10 mg mL1

6.1  1016 mol

–

[28]

TMB-d, 3,3u,5,5u-tetramethylbenzidine dihydrochloride.

2.3 DNA separation and amplification Capillary electrophoresis (CE) has been proven to be a powerful technique for fast and automatic DNA separation when hyphenated with FIA as the sample introduction front end [30]. This coupling scheme has been exploited extensively. Wang et al. proposed a compact microchip-based CE system for DNA separation using laser-induced fluorescence (LIF) detection and incorporating a liquid-core waveguide [31]. Automatic sample introduction was readily realized in an SIA system through a modified split-flow interface, which allowed the release of gas bubbles thus improving the stability of the system. In order to facilitate the integration and miniaturization of this chip-based SIA-CE analysis system, a light emitting diode (LED), rather than a laser beam, was further adopted as the excitation source with lock-in amplifier to enhance the signal-to-noise (S/N) ratio [32]. The feasibility of this compact manifold has been demonstrated by the successful separation of 11 components of an F174 HaeIII DNA digest sample. All the 11 components in the sample were effectively separated in 400 s with satisfactory resolution, with an S/N ratio comparable to that obtained by employing an SIA–CE system with LIF detection. The vast potential of FIA and SIA systems in fluidic manipulation also provides automatic sample preparation protocols for DNA amplifications. At this point, Belgrader et al. [33] developed a reusable flow-through PCR system for

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continuous monitoring of infectious biological agents. They employed an SIA system as the sample introduction front-end, which could be used repetitively to carry out the sequential analysis of samples. This provided great advantages over conventional PCR schemes with disposable reaction tubes for single use. In addition, much lower sample consumption was achieved, i.e., a reagent volume of less than 10 mL resulted in significant reduction of the running costs. A continuous flow PCR amplification system consisting of an SIA sampling processor, a microfluidic PCR chip and a 3-temperature-zone heating block was recently described by Liu et al. [34]. The system was capable of performing continuous PCR amplification with a low carryover between neighboring/ stacking sample zones. In order to reduce the dispersion and flow resistance of the reaction solution in the channels of the microchip, which could be considered to be the main source of cross contamination, the same research group designed a spiral-channel flow-through PCR microchip reactor. This system allowed the successful continuous amplification of seven samples in 1 h without cross contamination between samples [35].

3. ASSAYS OF PROTEINS, PEPTIDES AND AMINO ACIDS The analysis of proteins, peptides and amino acids can provide abundant information for diagnosis of various diseases, and thus their quantitative assays are of great importance in biological investigations as well as in clinical applications. Flow-based techniques have gained extensive attention and become much more popular in the fields mentioned herein attributed to the simple instrumentation required, ease of operation, reliability and low running costs.

3.1 Quantitative assay protocols It has been well documented that the weak resonance light scattering (RLS) intensity of some dyes could be significantly enhanced in the presence of trace amounts of proteins. These dyes include Bromothymol Blue [36], Amide Black-10B [37], Eriochrome Black T [38] and Biebrich Scarlet [39]. Various FIAbased automatic procedures for protein quantification have been developed recently. As compared to the conventional analytical procedures, these procedures provide much faster and inexpensive assays of total protein species in biological samples such as urine and serum. Chemiluminescence is a very sensitive and selective detection technique, which is most suitable for hyphenating with FIA. This detection technique has been recently utilized extensively for the direct determination of amino acids prior to their separation [40–43]. The most important key point to the success in achieving satisfactory selectivity in chemiluminescence detection is the choice of an appropriate reaction system and the manipulation of the chemical conditions in order to yield a response from the species of interest only. Costin et al. [40] proposed an FIA chemiluminescence methodology for selective detection of proline, histidine, tyrosine, arginine, phenylalanine and tryptophan in the

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presence of other amino acids. Selectivity was achieved by the application of a number of chemiluminescence reaction systems in addition to the manipulation of the reaction conditions where a certain amino acid gives rise to a response as a result of a particular reaction only. This approach offers significant advantages over conventional methods as derivatization, separation or extraction are not required, which dramatically reduces the analysis time. On the other hand, however, the employment of a series of chemiluminescence reactions obviously makes the overall analysis much more complicated. The chemiluminescent reactions between some amino acids and carbonyl functional groups of humic acids were used in an FIA system for the selective determination of glycine and arginine [41]. Considerable selectivity for these two amino acids in the presence of other amino compounds was achieved with detection limits of 0.20 and 0.25 mg L1 for glycine and arginine, respectively, along with a sampling frequency of 115 h1. A fluorescent derivative of albumin participates in a chemiluminescence reaction with peroxyoxalate with imidazole as the catalyst. By conducting this chemiluminescence reaction in an FIA system, an assay procedure for albumin was developed by employing a micellar medium as carrier [44]. A detection limit of about 0.1 fmol for albumin was achieved. Electroanalytical systems have been widely employed for the assay of macrobiomolecules in different flow analysis setups. Based on the electrocatalytic oxidation of cysteine at a pretreated platinum electrode, a selective FIA method for the biamperometric determination of this compound in amino acid mixtures and human urine samples was developed [45]. This method was characterized by a sampling frequency of 180 h1 and was applied to the determination of cysteine in real samples without any sample pretreatment. The assay of cysteine has also been performed by employing an FIA amperometric detection system based on the reaction of amino acids with chloramine-T [46]. A linear calibration graph of up to 10 mg cysteine mL1 was obtained, along with a sampling frequency of 220 h1. Nanjo et al. [47] have proposed recently an FIA system with an enzyme reactor and a hydrogen peroxide electrode for the measurement of fructosyl amino acids and fructosyl peptides in protease-digested blood samples. In their studies, fructosyl–amino acid oxidase and two fructosyl–peptide oxidases were covalently immobilized onto an inert support in the enzyme reactor. The proposed FIA system responded linearly to the concentration of fructosyl valine over the dynamic range 7.8  106–5.8  104 mol L1. The authors also proposed a similar FIA system, comprised of an electrochemical detector with fructosyl–peptide oxidase reactor and a flow-through spectrophotometer for the simultaneous measurement of glycohemoglobin and total hemoglobin in blood cell [48]. The hyphenation of FIA sample processing with high performance liquid chromatography (HPLC) provides promising potentials for both the elimination of interfering sample matrices and separation of the target species. An FIA system coupled to an HPLC system has been successively employed for detecting low molecular mass advanced glycation end-peptides (AGE-P) in samples from 126 diabetic patients, 54 normal controls and 20 diabetic mice [49]. The variance

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coefficients for intra-assay and inter-assay were 1.2% and 6.3%, respectively, which suggested that the AGE-P assayed by FIA provided a better precision and recovery compared to the protocols based on batch enzyme-linked immunosorbent assays (ELISA) and fluorescence spectrometry. As is well documented in the literature, FIA is suitable for coupling with almost any type of a detection system. The performance of FIA systems coupled to electrospray ionization mass spectrometry (ESI–MS), tandem mass spectrometry and electrospray ionization high-field asymmetric waveform ion mobility mass spectrometry (ESI–FAIMS) has been investigated for the suitability of these techniques for the determination of underivatized amino acids [50]. The experimental results have shown that ESI–FAIMS–MS offered improved sensitivity and significantly better S/N ratio when compared to ESI-MS. This was mainly due to the elimination of the background noise and the partial or complete resolution of all potential isobaric overlaps arising from amino acids. These results suggested that ESI–FAIMS–MS should be the preferred method for the quantitative analysis of proteinogenic amino acids in real samples. A sensitive procedure for the quantification of total protein in human serum involving SIA sampling and fluorometric detection, based on the rapid reaction between fluorescamine and primary amino acids, was proposed [51]. A few microliters of sample and fluorescamine solutions were mixed and the reaction of proteins with fluorescamine gave rise to a blue-green–fluorescent derivative, which was subsequently excited at 400 nm and the fluorescence was monitored at 470 nm. By loading 5.0 mL of sample and 4.0 mL of 0.075% (m/v) fluorescamine solution, a linear calibration graph was obtained within 0.3–12.5 mg mL–1 along with a substantially improved detection limit of 0.1 mg mL–1 as compared to 10.0 mg mL1 for the conventional manual procedure based on the same reaction system. As the third generation of flow analysis systems, an LOV analyser provides vast potential in bioassays attributed to its unique structural characteristics. In one of the recent studies, protein coated Sepharose beads were introduced into the flow cell of an LOV, where the beads were trapped by the tip of an optical fiber and the changes of spectral properties on the beads surface was monitored in situ. A label dilution protocol was thus developed to discriminate between the selective and non-selective bindings [52] which not only provided a protocol for monitoring bioligand interactions in real time, but also presented a sensitive method for the determination of low levels of analytes of interest in complex matrices in immunoassays. As a model analyte, the determination of immunoglobulin G (IgG) was performed with a detection limit of 470 ng. Based on a similar principle, i.e., selective capture and release of the analyte of interest on an appropriate stationary phase, micro-affinity chromatography (m-AC) and micro-bead injection analysis spectroscopy (m-BIAS) have been developed. The detection modes of the two techniques are quite different from each other. Both techniques have been recently applied to the determination of IgG as a model analyte in the same LOV system [53]. The beads were retained up-stream in the flow cell of the LOV system. The absorbance of the eluted analyte was monitored post-column in the m-AC procedure, while the spectral

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changes of the beads’ surface were detected in m-BIAS mode. When employing a longer light path in the absence of light scattering m-AC exhibited higher sensitivity compared to that of m-BIAS, i.e., the limit of detection of the m-AC technique for IgG was 5 ng mL1, and that of the m-BIAS technique was 50 ng mL1. The main analytical characteristics of flow-based techniques applied to quantitative assays of proteins, peptides and amino acids are summarized in Table 2.

3.2 Activity measurements The transient features of the signal recorded under thermodynamically nonequilibrium conditions in an FIA system are most suitable for measuring activitybased properties of certain biological species or compounds and reagents. A rapid FIA assay protocol for determining the activity of the purified catechol-Omethyltransferase (COMT) from porcine liver using electrochemical oxidation, fluorogenic derivatization, and fluorescence detection was described by Aoyama et al. [54]. It was demonstrated that the kinetic parameters obtained by using this FIA procedure were similar to those derived from an HPLC system but the FIA approach offered a much higher sample throughput. Recently, Staggemeier et al. [55] developed an FIA procedure based on coupling a linear pH gradient system and a dynamic surface tension detection unit (DSTD) for protein surface activity measurements (Figure 3). This system allowed high sample throughput screening of protein surface activity at the air/ liquid interface as a function of pH. This method not only provided an innovative approach for probing the pH-induced conformational changes of proteins by exploring surface tension measurements, but represented also a further advancement of conventional methodologies based on spectrometric measurements. This research group also developed an FIA manifold incorporating in parallel a multi-dimensional DSTD system and a UV-VIS diode array absorbance detector [56]. The system was used specifically for studying the effects of chemical denaturants, such as urea, guanidinium hydrochloride, and guanidinium thyocyanate, on the surface activity of globular proteins at the liquid-air interface.

3.3 Separation of biomolecules During the last decade, quite a few investigations have been directed towards the hyphenation of FIA with CE. Fang’s and Karlberg’s groups have made significant contributions towards the development of this field independently [30,57]. Their pioneering work on the hyphenation of an FIA sample pretreatment front end with a CE separation system greatly enhanced both sample injection and separation efficiency. In addition, the reduced consumption of samples and reagents as well as the possibility to separate and analyse small molecules in complex matrices opened promising avenues for applications in biochemistry.

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Applications of flow-based techniques for quantitative assays of proteins, peptides and amino acids

Analyte

Detection mode

Linear range

Detection limit

Sampling frequency

References

Protein

Rayleigh light scattering Rayleigh light scattering

7.0–70.0 mg mL1

3.75 mg mL1

26 h1

[36]

0.50–32.00 mg mL1 for HSA 2.00–36.00 mg mL1 for BSA 7–36 mg mL1 for HSA 8–44 mg mL1 for BSA 0.005–18 mg mL1 for HSA 0.008–16 mg mL1 for BSA 1  108–1  105 mol L1

0.11 mg mL1 for HSA

–

–

0.85 mg mL1 for BSA

90 h1

[37]

0.882 mg mL1 for HSA 2.507 mg mL1 for BSA 5 ng mL1 for HSA

– 90 h1 –

– [38] –

7.8 ng mL1 for BSA

–

[39]

4  109 mol L1 for proline 1  108 mol L1 for tyrosine 4  107 mol L1 for histidine

–

[40]

Protein

Protein Protein

Amino acids

Rayleigh light scattering Rayleigh light scattering

Chemiluminescence

Jianhua Wang and Xuwei Chen

Table 2

Chemiluminescence

1–30 mg L1

Tryptophan

Chemiluminescence

L-cysteine albumin L-cysteine Cysteine Valine

Chemiluminescence Chemiluminescence Amperometry Amperometry Amperometry

Histidine

Amperometry

AGE-P Protein IgG IgG

Fluorometry Fluorometry Spectrophotometry Spectrophotometry

6.0  107– 3.0  105 mol L1 0.2–80 mg L1 1.02–12 mg L1 4  107–4  105 mol L1 0.2–10 mg mL1 7.8  106– 5.8  104 mol L1 7.0  106– 1.1  104 mol L1 0.01–10 mg mL1 0.3–12.5 mg mL1 0.1–1.0 mg mL1 0.1–0.4 mg mL1

115 h1

[41]

50 h1

[42]

0.1 mg L1 0.38 mg L1 1  107 mol L1 0.06 mg mL1 –

60 h1 180 h1 220 h1 –

[43] [44] [45] [46] [47]

1.4  106 mol L1

–

[48]

– 0.1 mg mL1 5 mg mL1 (mAC) 50 mg mL1 (mBIS)

– 40 h1 – –

[49] [51] [53] [53]

Life Sciences Applications

Amino acids

1  107 mol L1 for arginine 7  106 mol L1 for phenylalanine 2  106 mol L1 for trytophan 0.20 mg L1 for glycine 0.25 mg L1 for arginine 1.8  107 mol L1

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Pump Sample

Computer Injection Valve

30 µL/min

60 µL/min

Waste

pH meter

Solenoid Valve

Mixing Coil 30 µL/min

Po Pressure Sensor

pH Gradient System

Capillary Sensing Tip and Drop

Air Supply Air Burst Capillary (Pneumatic Detachment)

Drop Collection Vessel

Figure 3 Schematic diagram of the FIA-pH-DSTD instrument configuration. The pressure sensor, connected to the tubing with a sidearm, measures the differential pressure across the liquid/air interface with respect to atmospheric pressure, Po, of the forming drops. Reprinted from [55]. Copyright (2005), with permission from the American Chemical Society.

When an SIA system is employed, the discrete zones of samples and/or reagents disperse into each other, thus resulting in a reproducible zone penetration. Consequently, the reaction product is formed in a well-defined area of concentration gradients and this provides reproducible analytical results, thus making SIA systems suitable candidates for on-line sample pretreatment such as pre-column derivatization. An FIA-based split-flow sample introduction system was developed and coupled to a CE system through a falling-drop interface [58]. A sampling throughput of up to 144 h1 was achieved along with a 2% carryover and an RSD of 3.2% by continuously introducing a series of 30 mL sample solutions containing a mixture of fluorescein isothiocyanate (FITC)-labeled amino acids. The same research group developed later an SIA micro-chip-based CE system for the separation of amino acids which incorporated a split-flow sampling unit, similar to the one mentioned above and integrated onto the micro-chip [59]. Sequential introduction of a series of 3.3 mL sample solutions containing a mixture of FITC-labeled amino acids gave rise to a carryover of 2.5% at a sample throughput of 48 h1. Baseline separation of FITC-labeled arginine, phenylalanine, glycine and FITC in sodium tetraborate buffer was achieved within 8–80 s. An on-column polymer-imbedded graphite inlet electrode for CE coupled on-line to an FIA system by using a poly(dimethylsiloxane) interface was proposed [60]. The electrode consisted of a conductive polyimide/graphite imbedded coating immobilized onto the CE column inlet. This integrated

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electrode gave the same separation performance as a commonly used platinum electrode. The on-line FIA–CE system was used with electrospray ionization-time of flight-mass spectrometric detection. The authors validated this hyphenation technique by a successful separation of three peptides (methionine–enkephalin, neurotensin, and substance P) in an electrolyte consisting of 50% formic acid/ ammonia and 50% acetonitrile. The on-line coupling of SIA and CE via an in-line injection valve for automated derivatization of amino acids and peptides has recently been described [61]. Dichlorotriazinylaminofluorescein served as the derivatization agent, enabling sensitive laser-induced fluorescence detection of the derivatives. When using des-tyr(1)-[met]-enkephalinamide as the model analyte, on-line electrophoretic analysis was achieved. Glycine was selected as the internal standard in order to correct for variations in reaction time and filling of the injection loop. For enkephalin, good reproducibility, linearity and a favorable limit of detection of 30 ng mL1 were achieved. Wu et al. [62] reported the successful hyphenation of mSIA–LOV with a CE system, where the LOV acted as the sampling ‘‘front end’’ for the CE setup, and this integrated mSIA–LOV–CE system was used for in situ protein derivatization [63], as illustrated in Figure 4. All the necessary micro-fluidic manipulations such as sampling, fluorogenic labeling, and CE capillary regeneration were automatically performed by the mSIA–LOV unit. On-line fluorogenic derivatization of Islet proteins (insulin, proinsulin and c-peptide) was carried out with fluorescamine which was followed by successful CE separation and fluorometric detection. The RSD values for peak area, electro-migration time and peak height using 3.45 mmol L1 insulin injections were 1.3%, 0.5% and 2.8%, respectively. A miniaturized sequential affinity chromatography within an LOV system was developed for the separation of mouse IgG, chicken IgG and bovine serum albumin. An automatically renewable micro-column was integrated into the LOV module used for separation and quantification of biomolecules on Sepharose Protein A beads by absorbance measurements at 280 nm. This setup reduced greatly the sample and reagent consumption and the total assay time. In addition, a favorable limit of detection of 6.0 ng mL1 for mouse IgG was obtained [64]. Ogata et al. [65] reported on two other types of LOV–BIA geometries for investigating the automated selective capture and release of biotin-containing conjugates on immobilized streptavidin. The capturing and releasing procedure were monitored on-line by UV/VIS spectrometry and the dissociation procedure was simultaneously monitored by ESI–MS. The LOV–ESI–MS instrument was also used for repetitive assays of lysosomal beta-galactosidase in human cell homogenates. Fast analysis in 4.5 min for a full cycle and robust operation in 60 repetitive analyses were demonstrated thus making the transfer of the LOV–ESI–MS technology into clinical practice very promising. A similar procedure was used for the simultaneous measurement of the affinities of multiple ligands to proteins [66]. In this automated LOV mode, 1 mg of protein was sufficient for 35 repetitive analyses and the equilibrium dissociation constants (Kd) could be determined rapidly in the range of 105–107 mol L1.

574

Anode

Waste Sample To Isolation Valve Flow-through Port

LOV PMT

Spectrometer

Waste

2

Objective Lens

CE Buffer

1

6 5

Optical Fiber

3

6-way Selector Valve

4

Capillary Cathode

Reagent Epiluminescence Microscope Syringe Pump

0.1M NaOH

UV Source

0.1M Phosphoric UV Source Acid

Holding Coil

CE Buffer Reservoir LOV

Figure 4 Schematic diagram of an LOV–CE derivatization system for peptides. Reprinted from [63]. Copyright (2003), with permission from the Royal Society of Chemistry.

Jianhua Wang and Xuwei Chen

Spectrometer

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3.4 Protein immobilization Immobilization chemistry is of key importance for the successful production of selective supports. Using an LOV–BIA manifold, Ruzicka et al. [67] studied the immobilization reactions of six proteins (albumin, ovalbumin, lysozyme, human IgG, ribonuclease A and cytochrome C) on the surface of agarose beads by measuring the rate and yield of the coupling reactions. By exploiting the BIA technique, the currently recommended protocols for reductive amination were shortened from several hours to only a few minutes. The leakage of immobilized ligands was measured by in situ direct spectrometric monitoring of the captured beads. These results suggested that BIA spectrophotometry was a useful tool for quality control of agarose-based chromatographic supports. It could also be used for the optimization of a wide variety of immobilization chemistries, used for synthesis of chromatographic supports, immobilization of enzymes, and derivatization of biosensing surfaces. The real-time monitoring of protein immobilization by using this protocol resulted in the surprising finding that current immobilization protocols were far from optimal.

4. IMMUNOASSAYS Flow-based immunoassay protocols, especially FIA immunoassay (FIA–IA) and SIA immunoassay (SIA–IA) have proven to be very useful in eliminating the drawbacks of conventional immunoassay schemes, which are usually timeconsuming and labor-intensive. Due to their unique characteristics such as minimized sample consumption, capability of sample pretreatment, and the ease of automation when high sample throughput is pursued, FIA–IA and SIA–IA have been extensively employed in various fields, among which life sciences applications have attracted extensive attention. Both methodologies are very suitable for hyphenating with various detection techniques, such as electrochemical methods, fluorometry, chemiluminescence methods and spectrophotometry. The flow-based immunoassay with electrochemical detection is one of the most developed methodologies. So far, the majority of the amperometric flow immunoassay systems have involved an immunoreactor, in which the antibody-antigen incubation step and the enzyme reaction take place. Another separate amperometric detector is employed for the oxidation or reduction of the enzyme-generated electroactive product at the surface of an appropriate electrode. Therefore, the design and implementation of a unique sensing surface for facile ligand functionalizations and biospecific interactions are very critical. Screen-printed electrodes have been extensively explored in recent years for bimolecular immunoassay [68–72], due to their low price and satisfactory reproducibility. With the aim of simplifying the flow-based enzyme immunoassay systems with amperometric detection, a promising approach has been developed by combining the immunoreactor and the detection unit into a single device by

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immobilizing the immunoreagent directly onto the electrode surface, which serves as an immunosensor. Valat et al. [73] have reported on a protein A-based FIA–IA immunosensor for enzyme immunoassay of rabbit IgG and mouse IgG. It has been demonstrated that this immunosensing system can be used repetitively for 30 assay cycles. It is also disposable whenever necessary. An immunosensor for rapid separation-free determination of carcinoembryonic antigen (CEA) in human serum was developed by co-immobilizing thionine and horseradish peroxidase (HRP)-labeled CEA antibody on a glassy carbon electrode (GCE) through covalently binding with glutaraldehyde (GA) linkage. This immunosensor showed good accuracy, acceptable storage stability and favorable precision when employed in an FIA system. After the system had been optimized, a detection limit of 0.1 ng mL1 for CEA was achieved [74]. Chemiluminescence has become a very attractive detection technique in FIA–IA applications because of its simple instrumentation, very low detection limit and wide linear dynamic range. HRP is commonly used in chemiluminesence detection by catalyzing the oxidation of luminol by hydrogen peroxide (H2O2). A chemiluminesence FIA system has recently been employed in the immunoassay of Estriol [75], a-fetoprotein [76,77], 17 b-estradiol [78] and CEA [79]. The performance of FIA–IA and SIA–IA with chemiluminesence detection in the determination of a-amino acids with an immunoreactor consisting of a flow cell packed with immobilized haptens was investigated by Silvaieh et al. [80]. The experimental results indicated that better repeatability and higher sampling frequency were obtained by SIA–IA. The corresponding detection limits were 1.01 ng mL1 for the FIA–IA system and 0.29 ng mL1 for the SIA–IA system, which were further improved to 0.22 ng mL1 and 0.036 ng mL1, respectively, by employing stopped-flow mode. Although bioluminescence is not employed as extensively as chemiluminescence in flow-based immunoassay systems, it is indeed a very useful tool for some specific purposes and certain analytes of interest. Ho and Huang [81] recently described an FIA–IA system based on bioluminescence with liposomal aequorin as the label, which utilized the binding-site-directed immobilization of anti-biotin antibodies in a microcapillary using protein A. This system allowed the detection of 50 pg of biotin, which was a 60-fold improvement in sensitivity as compared to a similar FIA–IA system with fluorescence detection. Among the various flow-through immunosensors, quartz crystal microbalance (QCM) immunosensors have started to play an important role, which they deserve. QCMs are developed by the immobilization of antigen or antibody onto the surface of a piezo-electric material. It is crucial to obtain satisfactory immobilization to ensure high sensitivity and stable response in practical QCM applications. Four types of approaches including physical immobilization, and three chemical approaches (i.e., thioamine thiolation and the use of periodateoxidized dextran-modified thioamine or a thiol-gold chemisorption-based self-assembled monolayer (SAM)) have been employed by Liu et al. [82] to immobilize human serum albumin (HSA) onto the surface of a QCM. The performance of these QCMs has been investigated in an FIA–IA system. It was observed that all four methods had lead to comparable detection limits of the

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QCM–FIA immunoassay. However, the SAM-based approach generated the largest frequency shift and also yielded the largest linear detection range. This indicated that the use of thiolated long-chain fatty acid forming a SAM might potentially be of greater interest as a protein immobilization method in QCM– FIA applications. A conducting polymer entrapment (CPE) method for immobilizing immuno-proteins on QCM has been also proposed by the same research group [83]. A higher frequency shift compared to that obtained by physical immobilization has been achieved. Fluorescence has been a key immunoassay detection technique for many years, yielding a large variety of procedures in this field. A simple and specific FIA–IA procedure for the detection of CEA by timeresolved fluorescence has been developed by Yan et al. [84] and it has exhibited higher sensitivity than conventional immunoassays can offer. This procedure was based on a sandwich immunoassay format involving the immobilization of a monoclonal antibody into an immunoaffinity column acting as an immunoreactor in the corresponding FIA–IA system. The cleaved solution was detected by time-resolved fluorescence after the reaction between the immunocomplex in the immunoaffinity column and the enhancement solution that was used to cleave the Eu-labels from the immunocomplex. Serum samples containing CEA have been detected in a linear range 2.5–100 ng mL1 along with a limit of detection of 1.0 ng mL1. The analysis of a large number of human serum samples showed good agreement with the results obtained by an alternative electrochemiluminescence immunoassay approach. The proposed time-resolved fluorescence FIA–IA method could be further developed for fast clinical detection of serum containing different levels of CEA. Although the detection sensitivity of spectrophotometry is somewhat lower as compared to other spectrometric techniques, it is frequently employed in immunoassays and is indispensable for solving some specific problems. A spectrophotometric immunoassay protocol was developed for vitellogenin (Vg) [85]. This method utilized an SIA system equipped with a jet ring cell, in which the immunoassay was conducted by using primary antibody-immobilized on Sephadexs beads and an HRP-labeled secondary antibody. The major drawback of this system was that the time required for the immunoreaction, i.e., 3 h, was somewhat too long for a quantitative assay. This might be attributed to the slow colour-development reaction. In order to handle this issue, an SIA-based chemiluminescence procedure for the determination of Vg using magnetic microbeads coated with an agarose gel instead of Sephadexs beads was developed [86]. The assay time was significantly shortened, though 20 min were still required for completing the assay. Most of this time was needed for the incubation of the immunoreaction system because of the slow diffusion of Vg or the secondary antibody into the agarose gel coated on the magnetic microbeads. It was recently discovered that magnetic microbeads coated with polylactic acid facilitated the rate of the immunoreaction [87,88]. Thus, a rapid and sensitive sandwich SIA-based immunoassay for the determination of Vg was developed. The SIA system consisted of a syringe pump, a multi-position valve and a flowthrough immunoreaction cell equipped with a magnet and an amperometric

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detector. Magnetic microbeads with an anti-Vg monoclonal antibody (primary antibody) immobilized on them were used as a solid support. After the primary antibody-immobilized magnetic beads were introduced and trapped in the immunoreaction cell, a Vg sample solution, an alkaline phosphatase (AP)-labeled anti-Vg polyclonal antibody (secondary antibody) solution and a p-aminophenyl phosphate (PAPP) solution were sequentially introduced into the immunoreaction cell. Vg was determined by the electrochemical detection of p-aminophenol (PAP), an enzymatic product of PAPP by the action of AP of the secondary antibody. A solution containing PAP, which was generated in the immunoreaction cell was transported to the amperometric detector where the oxidation current of PAP flowing through the working electrode was measured. The detection limit of the immunoassay was about 2–3 mg L1. The entire reaction time required for this system was reduced to less than 15 min. Two sampling approaches for flow-based chromatographic competitive binding immunoassay, i.e., the simultaneous and sequential injection methods, were investigated by Nelson et al. [89]. Both techniques used a column with a limited amount of antibody, subjected to a perfusion of sample and a labeled analyte analog. In the simultaneous injection mode, the sample and labeled analog were introduced at the same time into the column, while in the sequential injection mode the sample was injected first, followed by that of the analog. This resulted in different analytical characteristics of these two approaches. This study used chromatographic theory and data previously obtained by injecting HSA into an anti-HSA antibody column to compare the response, detection limit, linear range and sensitivity of these methods. Under equivalent conditions, it was found that the sequential method provided a lower limit of detection. The simultaneous mode offered a broader linear range and a higher upper limit of detection. In flow-based heterogeneous immunoassays, a solid support is generally used to immobilize either the antibody or antigen, thus permitting the separation of free fractions from bound immunocomplexes. Usually, a relatively long time is needed for regeneration of the solid support, which results in a lower sampling frequency. At the same time, some irreversible changes of the surface characteristics of the immunoreactor caused by its repetitive use might deteriorate the reproducibility. Therefore, carrying out the immunoassay by appropriately handling of the solid support material is of great importance. Renewable surface techniques seem to be the most suitable approach. An SIA bead-based immunoassay system has been developed by Hartwell et al. [90] for the determination of hyaluronan (HA). The main purpose of this study was to automate the immunoassay by ensuring precise delivery of micro-volumes of reagents and precise timing of the incubation and washing steps. These operations were achieved by computer control of the flow system’s bi-directional syringe pump. The manifold was designed with the aims of (i) reducing back pressure from beads that acted as solid surfaces for immobilization of the target substance; (ii) reducing dispersion and dilution of the reagents during incubation and (iii) maximizing the signal while minimizing the incubation time. This was

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achieved by introducing air segments to separate the reagent zones from the carrier stream and by using a suitable sensitive detector, i.e., an amperometric sensor. The amount of HA was determined using competitive ELISA-based technique where immobilized HA and HA in solution competed to bind with a fixed amount of biotinylated-HA-binding proteins (b-HABPs). Upon separation of the two phases, anti-biotin conjugated with enzyme and a suitable substrate were introduced to follow the binding reaction of the immobilized HA and b-HABPs, whose degree of binding was indirectly proportional to the amount of HA in solution. Varnum et al. [91] described an enzyme-amplified protein microarray and a fluidic renewable surface fluorescence immunoassay for botulinum neurotoxin detection using high-affinity recombinant antibodies. By employing the renewable surface technique, the analysis time was reduced to less than 10 min, while at the same time the sensitivity was found to be somewhat lower compared to the conventional batchwise ELISA. An SIA renewable surface heterogeneous fluorescence immunoassay system with chip-based micro-flow-through cell has also been developed by Zhu et al. [92] for the determination of human IgG in serum. Immobilized antibody was prepared by conjugation of sheep anti-human IgG antibody to protein A coated Sepharoses CL4B beads. FITC labeled anti-human IgG antibody was used as the second antibody. The immobilized antibody beads, serum and the second antibody were sequentially injected into the chip-based micro-flow-through cell where a sandwiched antibody–antigen conjugate with fluorescence probe was formed and the fluorescence intensity was measured in the cell using optical fibers. After the measurement, the beads were discharged and the cell was ready for the next operation cycle. A detection limit for IgG of 0.1 mg L1 was achieved at a sample throughput of 11 h1. RSDs of 1.7% and 5.2% were obtained for inter-day and intra-day determinations of serum samples containing 3.9 mg L1 IgG. As the third generation of flow analysis techniques, the unique configuration of LOV is an ideal platform for BIA and provides vast potential for facilitating renewable surface operations, which have been one of the most important issues in immunoassay development. Carroll et al. [93] have reported on a novel analytical method for the detection and study of GAD65 autoantibodies, which have been implicated in the onset of Type 1 diabetes. There is a clinical need for a rapid and automated assay of GAD65 autoantibodies. The work mentioned above has focused on exploiting the advantages of BIA for ELISA in an LOV system. The BIA ELISA scheme is a microscale technique that uses enzyme labeled secondary antibodies to detect the capture of target antibodies on immobilized antigen in the flow cell of the LOV manifold. A detection limit of 20 ng mL1 for GAD65 monoclonal antibody 144 compares favorably with the sensitivity and precision of a standard ELISA currently employed to detect GAD65 autoantibodies. Compared to the standard ELISA protocol, BIA ELISA offers a significantly reduced assay time and complete automation of solution handling and detection. Table 3 summarizes the characteristics of selected flow-based immunoassay applications.

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Table 3 Applications of flow-based immunoassay systems Detection mode

Linear range

Detection limit

References

Alpha-fetoprotein

Amperometry

Alpha-fetoprotein Alpha-fetoprotein Interleukin-6 Carcinoembryonic Carcinoembryonic Carcinoembryonic Carcinoembryonic IgG IgG

Chemiluminescence Chemiluminescence Amperometry Amperometry Amperometry Chemiluminescence Time-resolved fluorometry Amperometry Amperometry

5–20 ng mL1 20–150 ng mL1 5.0–100 ng mL1 2.0–75 ng mL1 5–100 ng L1 0.50–25 ng mL1 0.5–3.0 ng mL1 3.0–167 ng mL1 1.0–25 ng mL1 2.5–100 ng mL1 30–700 ng mL1 –

Human IgG Estriol 17 beta-estradiol Biotin D-phenylalanine

Fluorometry Chemiluminescence Chemiluminescence Bioluminescence Chemiluminescence

0.3–7.0 mg L1 10.0–400 ng mL1 10.0–1,000 ng mL1 1  1011–1  103 mol L1 –

HSA Vitellogenin Vitellogenin Vitellogenin Hyaluronan Neurotoxin GAD65 autoantibodies

Quartz crystal microbalance Spectrophotometry Chemiluminescence Amperometry Amperometry Fluorometry Spectrophotometry

0.01–05 mg mL1 7.8–125 ng mL1 2–100 ng mL1 0–500 g mL1 1–5,000 ng mL1 – –

2 ng mL1 – 2.7 ng mL1 0.5 ng mL1 1.0 ng L1 0.22 ng mL1 0.1 ng mL1 0.5 ng mL1 1.0 ng mL1 3 ng mL1 0.02 mg mL1 (mouse IgG) 0.2 mg mL1 (rabbit IgG) 0.1 mg L1 5.0 ng mL1 3.0 ng mL1 50 pg 1.01 ng mL1 (FIA mode) 0.29 ng mL1 (SIA mode) 0.01 mg mL1 5 ng mL1 2 ng mL1 2–3 ng mL1 1 ng mL1 1.4 pg mL1 20 ng mL1

[68] – [76] [77] [69] [70] [74] [79] [84] [72] [73] – [92] [75] [78] [80] [81 – [82] [85] [86] [87,88] [90] [91] [93]

antigen antigen antigen antigen

Jianhua Wang and Xuwei Chen

Analyte

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5. ENZYMATIC ASSAYS On-line enzymatic reactions taking place in flow analysis systems have been widely employed in order to facilitate routine biochemical analyses and applications in biocatalysis. FIA and SIA enzymatic assay approaches are excellent choices for bioassays because of the unique characteristics of the flow systems including low sample and reagent consumption and thus reduced analysis costs, ease of operation, as well, as fast analysis.

5.1 Heterogeneous enzymatic assays based on microreactors Most of the flow immunoassay systems use enzymes immobilized on microbeads or on the interior surface of microfluidic channels, whilst some employ dissolved enzymes in order to perform reactions in microfluidic systems. Flowbased analytical systems incorporating microreactors are generally characterized by outstanding repeatability and reproducibility, which can be attributed mainly to the elimination of the drawbacks of iterative batch mode operations. The characteristic features of FIA and SIA systems fit perfectly with the applications of enzymatic microreactors as described in a recent review article [94]. The most commonly used enzymatic microreactors prepared by the immobilization of an appropriate enzyme onto a suitable supporting material are used for the direct determination of biomolecules that act as substrates in enzymatic reactions. A large variety of detection techniques can be employed for monitoring the enzymatic reactions, e.g., amperometry, chemiluminescence and spectrophotometry. An FIA method for the determination of serine, using a mini-column containing immobilized serine dehydratase isolated and purified from rat liver, has been developed [95]. Ammonia produced from the enzymatic reaction was reacted with hypochlorite and phenol in alkaline medium yielding the blue indophenol anion, which was detected spectrophotometrically at 640 nm. A limit of detection of 0.01 mM along with a sample throughput of 25 h1 was achieved. The usefulness of enzyme microreactors for the determination of very low concentrations of amino acids has also been demonstrated recently with the determination of cysteine [96] and L-aspartate [97]. For the determination of cysteine, a rotating biosensor and stopped-flow technique were adopted in order to improve the detection sensitivity [96]. Nanjo et al. [48] described an enzymatic FIA method for rapid measurement of hemoglobin A(1c) (HbA1c). The FIA system was comprised of an electrochemical detector with a specific enzyme-reactor, i.e., a fructosyl-peptide oxidase (FPOX-CET) reactor, and a flow-through spectrophotometer for the simultaneous measurement of glycohemoglobin and total hemoglobin in blood cells. First, total hemoglobin was determined spectrophotometrically in digested samples and then the fructosyl valyl histidine (FVH) released from glycohemoglobin by the selective proteolysis was selectively determined using the electrochemical detector with the FPOX-CET reactor. This FIA

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system was automatically run at a sampling rate of 40 h1. The enzymatically determined HbA1c values deduced from the concentration ratio of FVH and total hemoglobin were closely correlated with the HbA1c values certified by the Japan Diabetic Society and the International Federation of Clinical Chemistry (IFCC). A miniaturized biosensor has been developed by Dutra et al. [98] for the determination of uric acid in biological fluids. The amperometric biosensor was prepared by using a carbon paste electrode modified with uricase from Arthrobacter globiforms and tetracyanoquinodimethane as the electron transfer mediator. When incorporated into an FIA system, it allowed 50 measurements per hour for uric acid in a range of 1–100 mmol L1 with a precision of 0.20% RSD. Among the various procedures for glucose determination, those based on enzymatic reactions have been widely employed. Generally, these procedures are based on the generation of hydrogen peroxide during glucose oxidation by glucose oxidase (GOD), which usually takes place in the immobilized microractorer incorporated into the flow manifold. The hydrogen peroxide thus generated can readily be detected by various techniques. An SIA renewable surface reflectance spectrophotometric system for the enzymatic determination of glucose in human serum samples has been developed by Wang et al. [99,100]. A built chip-based flow-through cell was used to trap the microbeads, which could readily be renewed for each analytical cycle. The analytical results agreed well with those obtained by the phenol-4aminoantipyrine method. An automatic flow procedure for the determination of glucose in animal blood serum using glucose oxidase with chemiluminescence detection and based on multicommutation was described by Pires et al. [101]. The flow manifold consisted of a set of three-way solenoid valves assembled to implement multicommutation. Glucose oxidase was immobilized on porous silica beads and packed in a minicolumn. The procedure was based on the enzymatic degradation of glucose, producing hydrogen peroxide, which oxidized luminol in the presence of hexacyanoferrate(III) and giving rise to chemiluminescence. The results were in agreement with those obtained by the conventional method (LABTEST Kit) at the 95% confidence level. Chen et al. [102] developed an amperometric FIA biosensor system for glucose assay where the biosensor consisted of a chitosan membrane from the carapace of the soldier crab where glucose oxidase was immobilized. The sensor signal was linearly related to glucose concentration with good sensitivity and reproducibility. A three-layer polydimethylsiloxane/glass microfluidic SIA system with stationary phase particles immobilized on one side of the channel wall was developed by Xu and Fang [103] for the chemiluminescence detection of glucose. A conventional SIA system was coupled directly to the microfluidic system. Hydrostatic delivery of the reagents was used to achieve efficient and reproducible sample introduction at 10 mL level. A detection limit of 10 mmol L1 glucose was obtained.

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5.2 Homogeneous enzymatic assays The capabilities of flow-based techniques for manipulating very small volumes of expensive enzymes also offers certain advantages in the use of soluble, rather than immobilized, enzymes in enzymatic assay systems. This approach facilitates the elimination or alleviation of typical drawbacks associated with the commonly used packed-bed or open tubular column enzyme reactors, such as fouling of the surface of the packed material, carryover effects, flow resistance, loss of binding sites or functional groups and the employment of harmful organic solvents during the immobilization process. A multisyringe flow injection analysis (MSFIA) manifold has been developed as a powerful tool for performing automated enzymatic assays in a renewable format using soluble enzymes [104]. The flow manifold is shown schematically in Figure 5. The MSFIA system involved four glass syringes connected in a block to Waste SV6

Off Autosampler On

HC

Off

SV5

On

KR On

RL W

SV1 SV2 SV3 SV4

PSM

Cobalt

Carrier

S1 S2

Luminol / NaOH

Enzyme

Off

S4

S3

Figure 5 Schematic diagram of the multisyringe FIA setup assembled for chemiluminescence determination of glucose at ultra-trace levels using soluble enzymes (S1–S4, syringe pumps; SV1–SV6, three-way solenoid valves; HC, holding coil; KR, knotted reactor; RL, reactor line; PSM, photosensor module; and W, waste). Reprinted from [104]. Copyright (2004), with permission from the American Chemical Society.

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the same step-by-step motor and coupled to three-way solenoid valves. This flow-based configuration was suitable for handling minute volumes of soluble enzymes. It also accommodated reactions with divergent kinetic and pH demands, which were used in the indirect chemiluminescence assay of glucose at ultra-trace levels. The procedure involved on-line glucose oxidase-catalyzed oxidation of beta-glucose in a homogeneous phase to beta-glucono-delta-lactone and hydrogen peroxide. Subsequently, the generated oxidant merged downstream with an alkaline zone of 3-aminopthalhydrazide and a metal-catalyst zone of Co(II) at a high flow rate aiming at warranting maximum light collection from the fast chemiluminescence reaction. For the enzymatic assay of glucose under optimal conditions, a sample throughput of 20 h1 and a detection limit of 72 mg L1 were obtained. The advent of LOV resulted in the construction of miniaturized SIA systems by integrating the sampling conduit and flow cell into a microfabricated compact structure mounted atop a multi-position selection valve. A higher sampling frequency is usually obtained in the LOV protocol since the sampling line present in conventional SIA systems has been eliminated through integration and miniaturization. This has been well demonstrated on enzymatic assays of glucose and ethanol [105]. Sampling frequency could be further increased by processing two sample injections simultaneously and by optimizing the assay protocol through flow acceleration in the LOV system [106], which is achieved by isolating the flow cell from the rest of the LOV system by turning the groove of the multiposition valve away from the flow cell port. Thus, the sample, reagent, and spacer of run #2 can be stacked into the holding coil, while the reacting mixture from run #1 is being monitored in the flow cell, as illustrated in Figure 6. The sampling frequency of this accelerated protocol is comparable to that of FIA and

Loading run #2 RUN #2

(a) HC

C S

RUN #1 Sp

R

(b)

RUN #2

HC

C

P

RUN #1 S

P R

Sp

P

Sending run #2 to flow cell

Figure 6 Accelerated mSI-LOV protocol (C, Carrier; S, sample; R, reagent; Sp, spacer; P, product; HC, holding coil; P, flow cell). (a) Stacking sample, reagent and spacer of run #2 into the holding coil during the stopped-flow measurement of run #1. (b) Sending the stacked zones of run #2 to the flow cell, to wash out the flow cell and start the measurement of run #2. Note that the extended volume of the spacer prevents intermixing of the sample/reagent zones of run #1 and run #2, and assists in washing out the flow cell. Reprinted from [106]. Copyright (2004), with permission from the Royal Society of Chemistry.

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the analysis time is reduced from 200 s required for traditional SIA format to about 30 s in the LOV protocol. By attaching a micro-reactor to one port of the LOV system to enhance mixing, enzyme kinetics and inhibition were investigated by the same research group using acetylcholinesterase (AChE) and antiotensin-converting enzyme (ACE) as model analytes [107]. The Michaelis constant (Km) for AChE and ACE obtained by this system agreed well with those reported in the literature. Ohgami et al. [108] proposed a microfluidic system for the analysis of the activities of glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT). The system consisted of a glass chip with a microelectrochemical L-glutamate sensor and a polydimethylsiloxane sheet with a Y-shaped micro-flow channel. Sample solution and substrate solution for the enzymes were introduced from two injection ports at the end of the flow channel and then mixed immediately by diffusion in the mixing channel. The enzyme activities were measured rapidly without any other reagents. The relationship between the slope of the response curve and the enzyme activity was linear in the range 7–228 U L1 for GOT and 9–250 U L1 for GPT, respectively.

6. CELLULAR ANALYSIS Recently, Ruzicka and coworkers have focused their research on flow-based renewable surface techniques for cell-based assays by immobilizing the cells of interest onto the surface of an appropriate bead material [109–112]. The real-time renewal of the cellular materials is usually performed by employing the flow design of a jet ring cell. The rapid renewal of the beads allows the refreshment of the inspected cells and thus each assay can be conducted on a fresh set of cells. This makes in situ acquisition of information concerning the cellular activities feasible. In order to investigate the real-time cellular glucose consumption, which could be used to ascertain the effects of a hypoxic event in cells, Schulz and Ruzicka [113] developed a micro-SIA–LOV (mSIA–LOV) system with an integrated microbioreactor for real-time in situ determination of glucose consumption by live cells. The adherent cells were cultured onto microcarrier beads and packed into a renewable microcolumn within the mSIA–LOV system. Glucose sensing was performed through the use of a two-step nicotinamide adenine dinucleotide (NAD)-linked enzymatic process. The course of the assay was monitored in real-time by measuring the absorbance of NADH at 340 nm. This microsequential assay based on plug/nozzle design had a linear dynamic range for glucose of 0.1–5.6 mmol L1. The mSIA–LOV system allowed the assay to be carried out using only 40 mL of the enzyme reagent and 3 mL of sample. The technique was tested on a murine hepatocyte cell line (TABX2S) adhered to Cytopores beads. Rapid cellular glucose consumption was facilitated by a high cell density, which allowed a large number of cells (104–105) to be retained in a very small volume of 3 mL. In turn, this cell density resulted in the rapid depletion of glucose from the cell medium over a short time period of less than 2 min. Based on the same principle, cellular lactate extrusion rate was also

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determined by the same research group using a similar mSIA–LOV setup. A linear dynamic range 0.05–1.00 mmol L1 was achieved and the measurement could be conducted within 30 s [114]. Extracellular acidification rate (ECAR) is a key parameter for cell activity used for the evaluation of the factors that alter metabolic functions, such as stimulants, inhibitors, toxins as well as receptor and non-receptor mediated events. By coupling the BIA technique, an exploratory study on ECAR measurement has been carried out by Erxleben et al. [115] in an LOV manifold. Two kinds of beads with different design were used in this investigation, Cytopores beads were used for cell culturing and trapped in the central channel of the LOV system, while Sephadexs beads were employed for covalently binding the indicator and they were retained in the flow cell. Hydrogen ions extruded from the cells were accumulated during a stopped-flow period and then detected by the change in absorbance of the pH indicator solution. The feasibility of this approach was demonstrated by measuring ECARs of the mouse hepatocyte cell line of TABX2S and the results agreed well with those obtained by using the Cytosensor system.

7. PERSPECTIVES The three generations of flow analysis techniques, i.e., FIA, SIA and LOV, have been well accepted as indispensable tools in the automation of analytical procedures, offering at the same time low sample and reagent consumption and high sampling frequency. Their potentials have been extensively exploited during the last decades, yet these techniques are far from being fully exploited so far. Although only very limited investigations have been directed towards flowbased cellular analysis, the integration of microbioreactors with live cells into flow systems has been demonstrated to be a very promising approach in this field. This not only provides an alternative for in situ investigation of cell activities, but also offers an attractive approach to adopting biological cells as the functional material in SPE for sample pretreatment. FIA and SIA have been proven to be powerful analytical tools for on-line sample pretreatment. The introduction of LOV has further enhanced the capabilities of flow-based techniques for clean-up of micro samples, which is of high importance in the analysis of biological samples. The development of labon-chip or micro-total analysis systems (mTAS) have attracted extensive attention. However, there is 3–6 orders of magnitude difference between the volumes used in mTAS and in conventional sample pretreatment systems and this has lead to the so-called ‘‘world-to-chip’’ interfacing problem, which has plagued the further development of the mTAS systems. The solution of this problem arising from the mismatch of the processed volume scales between the ‘‘world’’ and the chip has remained a challenge. At this point, LOV has provided a promising sample processing front end for mTAS systems. The assay of macrobiomolecules, such as DNA, peptides and proteins, in complex sample matrices has gained increasing interest. Thus, the potential of

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the on-line sample pretreatment capabilities of flow systems when coupled to BIA for matrix removal and analyte preconcentration or even for chromatographic separation by selecting appropriate sorbent materials and suitable eluents is large. This field obviously deserves further investigation.

ABBREVIATIONS m-AC ACE AchE AP b-HABPs m-BIAS CE CEA COMT CPE DSTD ECAR ELISA ESI-FAIMS FIA-IA FITC FPOX FVH GCE GOD GOT GPT HA HAS IFCC Km LIF PAP PAPP PCR RLS RSC S/N SAM SPE Vg

Micro-affinity chromatography Antiotensin-converting enzyme Acetylcholinesterase Alkaline phosphatase biotinylated-HA-binding proteins Micro-bead injection analysis spectroscopy Capillary electrophoresis Carcinoembryonic antigen Catechol-O-methyltransferase Conducting polymer entrapment Dynamic surface tension detection unit Extracellular acidification Enzyme-linked immunosorbent assays Electrospray ionization high-field asymmetric waveform ion mobility mass spectrometry Flow injection analysis immunoassay Fluorescein isothiocyanate Fructosyl-peptide oxidase Fructosyl valyl histidine Glassy carbon electrode Glucose oxidase Glutamic-oxaloacetic transaminase Glutamic-pyruvic transaminase Hyaluronan Human serum albumin International Federation of Clinical Chemistry Michaelis constant Laser-induced fluorescence p-aminophenol p-aminophenyl phosphate Polymerase chain reactions Resonance light scattering. Renewable separation column signal-to-noise ratio Self-assembled monolayer Solid phase extraction Vitellogenin

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REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

E.H. Hansen, Flow injection bibliography, http://www.flowinjection.com/ K. Grudpan, Talanta, 64 (2004) 1084–1090. J. Saurina and S. Herna´ndez-Cassou, Anal. Chim. Acta, 438 (2001) 335–352. C.E. Lenehan, N.W. Barnett and S.W. Lewis, Analyst, 127 (2002) 997–1020. A. Economou, Trend. Anal. Chem., 24 (2005) 416–425. M.P. Nandakumar, E.N. Karlsson and B. Mattiasson, Biotechnol. Bioeng., 73 (2001) 406–411. J. Ruzicka, C.H. Pollema and K.M. Scudder, Anal. Chem., 65 (1993) 3566–3570. J. Ruzicka and L. Scampavia, Anal. Chem., 71 (1999) 257A–263A. D.P. Chandler, B.L. Schuck, F.J. Brockman and C.J. Bruckner-Lea, Talanta, 49 (1999) 969–983. C.J. Bruckner-Lea, T. Tsukuda, B. Dockendorff, J.C. Follansbee, M.T. Kingsley, C. Ocampo, J.R. Stults and D.P. Chandler, Anal. Chim. Acta, 469 (2002) 129–140. J.W. Grate, C.J. Bruckner-Lea, A.E. Jarrell and D.P. Chandler, Anal. Chim. Acta, 478 (2003) 85–98. C.J. Bruckner-Lea, M.S. Stottlemyre, D.A. Holman, J.W. Grate, F.J. Brockman and D.P. Chandler, Anal. Chem., 72 (2000) 4135–4141. X.W. Chen, Z.R. Xu, B.Y. Qu, Y.F. Wu, J. Zhou, H.D. Zhang, J. Fang and J.H. Wang, Anal. Bioanal. Chem., 388 (2007) 157–163. J.H. Wang, Anal. Bioanal. Chem., 381 (2005) 809–811. X.W. Chen, J.H. Wang and Z.L. Fang, Talanta, 67 (2005) 227–232. G.R. Fang, G.W. Song, L. Li, H.J. Zhan and X.H. Li, Spetrosc. Spect. Anal., 22 (2002) 631–633. E.M. Caldarone and L.J. Buckley, Anal. Biochem., 199(1) (1991) 137–141. R.H. Yang, K.M. Wang, D. Xiao, K. Luo and X.H. Yang, Anal. Chim. Acta, 432 (2001) 135–141. K.A. Edwards and A.J. Baeumner, Anal. Chem., 78 (2006) 1958–1966. G.W. Song, G.R. Fang, S.F. Lu, Z.K. He and Y.E. Zeng, Chinese J. Anal. Chem., 28 (2000) 982–984. X.W. Chen, W.X. Wang and J.H. Wang, Analyst, 130 (2005) 1240–1244. Y.J. Ma, M. Zhou, X.Y. Jin, Z.Y. Zhang, X.L. Teng and H. Chen, Anal. Chim. Acta, 501 (2004) 25–30. M. Zhou, Y.J. Ma, X.Y. Jin, X.L. Teng, Z.Y. Zhang and H. Chen, Chinese Chem. Lett., 14 (2003) 1051–1053. H. Chen, M. Zhou, X.Y. Jin, Y.M. Song, Z.Y. Zhang and Y.J. Ma, Anal. Chim. Acta, 478 (2003) 31–36. L. Yi, H.C. Zhao, C.Y. Sun, S. Chen and L.P. Jin, Spec. Chim. Acta A, 59 (2003) 2541–2546. T. Perez-Ruiz, C. Martinez-Lozano, V. Tomas and J. Martin, Anal. Chim. Acta, 402 (1999) 13–19. J. Wang, L. Chen and M. Chicharro, Anal. Chim. Acta, 319 (1996) 347–352. J. Wang, M. Jiang and B. Mukherjee, Anal. Chem., 71 (1999) 4095–4099. F. Song, F. Zhou, J. Wang, N. Tao, J. Lin, R.L. Vellanoweth, Y. Morquecho and J. Wheeler-Laidman, Nucleic Acids Res., 30 (2002) e72. Z.L. Fang and Q. Fang, Fresenius J. Anal. Chem., 370 (2001) 978–983. S.L. Wang, Z.R. Xu, X.F. Fan and Z.L. Fang, Chinese J. Anal. Chem., 34 (2006) 145–149. S.L. Wang, X.F. Fan, Z.R. Xu and Z.L. Fang, Electrophoresis, 26 (2005) 3602–3608. P. Belgrader, C.J. Elkin, S.B. Brown, S.N. Nasarabadi, R.G. Langlois, F.P. Milanovich, Jr., B.W. Colston and G.D. Marshall, Anal. Chem., 75 (2003) 3446–3450. J.H. Liu, X.F. Yin, G.M. Xu, Z.L. Fang and H.W. Chen, Chem. J. Chinese Univ., 24 (2003) 232–235. J.H. Liu, X.F. Yin and Z.L. Fang, Chem. J. Chinese Univ., 25 (2004) 30–34. E. Vidal, M.E. Palomeque, A.G. Lista and B.S.F. Band, Anal. Bioanal. Chem., 376 (2003) 38–41. Y. Li, L.J. Dong, Y.H. Zhang, Z.D. Hu and X.G. Chen, Talanta, 71 (2007) 109–114. Y. Li, L.J. Dong, W.P. Wang, Z. Hu and X.G. Chen, Anal. Biochem., 354 (2006) 64–69. K.J. Tan, Y.F. Li and C.Z. Huang, Luminescence, 20 (2005) 176–180. J.W. Costin, P.S. Francis and S.W. Lewis, Anal. Chim. Acta, 480 (2003) 67–77. J. Michalowski and A. Kojlo, Talanta, 54 (2001) 107–113. Y.D. Liang and J.F. Song, J. Pharmaceut. Biomed. Anal., 38 (2005) 100–106. B.X. Li, Z.J. Zhang, M.L. Liu and C.L. Xu, Anal. Bioanal. Chem., 377 (2003) 1212–1216. L. Gamiz-Gracia, A.M. Garcia-Campana, F.A. Barrero and L.C. Rodriguez, Anal. Bioanal. Chem., 377 (2003) 281–286. C. Zhao, J.C. Zhang and J.F. Song, Anal. Biochem., 297 (2001) 170–176.

Life Sciences Applications

589

46 M.C. Icardo, O.A. Estrela, M. Sajewicz, J.V.G. Mateo and J.M. Calatayud, Anal. Chim. Acta, 438 (2001) 281–289. 47 Y. Nanjo, R. Hayashi and T. Yao, Anal. Sci., 22 (2006) 1139–1143. 48 Y. Nanjo, R. Hayashi and T. Yao, Anal. Chim. Acta, 583 (2007) 45–54. 49 Z.L. Sun, N.F. Liu, B.H. Liu and J.P. Wang, Clin. Chim. Acta, 313 (2001) 69–75. 50 M. McCooeye and Z. Mester, Rapid. Commun. Mass Spectrom., 20 (2006) 1801–1808. 51 X.W. Chen and J.H. Wang, Talanta, 69 (2006) 681–685. 52 A.D. Carroll, L. Scampavia and J. Ruzicka, Analyst, 127 (2002) 1228–1232. 53 Y. Gutzman, A.D. Carroll and J. Ruzicka, Analyst, 131 (2006) 809–815. 54 N. Aoyama, M. Tsunoda, K. Nakagomi and K. Imai, Biomed. Chromatogr., 16 (2002) 255–260. 55 B.A. Staggemeier, E. Bramanti, C. Allegrini, K.J. Skogerboe and R.E. Synovec, Anal. Chem., 77 (2005) 250–258. 56 E. Bramanti, C. Allegrini, M. Onor, G. Raspi, K.J. Skogerboe and R.E. Synovec, Anal. Biochem., 351 (2006) 100–113. 57 P. Kuban and B. Karlberg, Trend. Anal. Chem., 17 (1998) 34–41. 58 S.L. Wang, X.J. Huang, Z.L. Fang and P.K. Dasgupta, Anal. Chem., 73 (2001) 4545–4549. 59 Q. Fang, F.R. Wang, S.L. Wang, S.S. Liu, S.K. Xu and Z.L. Fang, Anal. Chim. Acta, 390 (1999) 27–37. 60 J. Samskog, S.K. Bergstrom, M. Jonsson, O. Klett, M. Wetterhall and K.E. Markides, Electrophoresis, 24 (2003) 1723–1729. 61 C.K. Zacharis, F.W.A. Tempels, G.A. Theodoridis, A.N. Voulgaropoulos, W.J.M. Underberg, G.W. Somsen and G.J. de Jong, J. Chromatogr. A, 1132 (2006) 297–303. 62 C.H. Wu, L. Scampavia and J. Ruzicka, Analyst, 127 (2002) 898–905. 63 C.H. Wu, L. Scampavia and J. Ruzicka, Analyst, 128 (2003) 1123–1130. 64 H. Erxleben and J. Ruzicka, Analyst, 130 (2005) 469–471. 65 Y. Ogata, L. Scampavia, J. Ruzicka, C.R. Scott, M.H. Gelb and F. Turecek, Anal. Chem., 74 (2002) 4702–4708. 66 Y. Ogata, L. Scampavia, T.L. Carter, E.K. Fan and F. Turebeka, Anal. Biochem., 331 (2004) 161–168. 67 J. Ruzicka, A.D. Carroll and I. Lahdesmaki, Analyst, 131 (2006) 799–808. 68 Z. Dai, S. Serban, H.X. Ju and N. El Murr, Biosens. Bioelectron., 22 (2007) 1700–1706. 69 K.Z. Liang and W.J. Mu, Anal. Chim. Acta, 580 (2006) 128–135. 70 J. Wu, J.H. Tang, Z. Dai, F. Yan, H.X. Ju and N. El Murr, Biosens. Bioelectron., 22 (2006) 102–108. 71 C.T. Hsu, H.H. Chung, H.J. Lyuu, D.M. Tsai, A.S. Kumar and J.M. Zen, Anal. Sci., 22 (2006) 35–38. 72 Q. Gao, Y. Ma, Z.L. Cheng, W.D. Wang and M.R. Yang, Anal. Chim. Acta, 488 (2003) 61–70. 73 C. Valat, B. Limoges, D. Huet and J.L. Romette, Anal. Chim. Acta, 404 (2000) 187–194. 74 Z. Dai, F. Yan, H. Yu, X.Y. Hu and H.X. Ju, J. Immunol. Methods, 287 (2004) 13–20. 75 S.H. Wang, L.Y. Du, S.L. Lin and H.S. Zhuang, Microchim. Acta, 155 (2006) 421–426. 76 Z.F. Fu, C. Hao, X.Q. Fei and H.X. Ju, J. Immunol. Methods, 312 (2006) 61–67. 77 J.H. Lin, F. Yan and H.X. Ju, Appl. Biochem. Biotech., 117 (2004) 93–102. 78 S.H. Wang, S.L. Lin, L.Y. Du and H.S. Zhuang, Anal. Bioanal. Chem., 384 (2006) 1186–1190. 79 J.H. Lin, F. Yan and H.X. Ju, Clin. Chim. Acta, 341 (2004) 109–115. 80 H. Silvaieh, M.G. Schmid, O. Hofstetter, V. Schurig and G. Gubitz, J. Biochem. Biophys. Methods, 53 (2002) 1–14. 81 J.A. Ho and M.R. Huang, Anal. Chem., 77 (2005) 3431–3436. 82 Y.C. Liu, C.M. Wang and K.P. Hsiung, Anal. Biochem., 299 (2001) 130–135. 83 Y.C. Liu, C.M. Wang, K.P. Hsiung and C.J. Huang, Biosens. Bioelectron., 18 (2003) 937–942. 84 F. Yan, J.N. Zhou, J.H. Lin, H.X. Ju and X.Y. Hu, J. Immunol. Methods, 305 (2005) 120–127. 85 N. Soh, H. Nishiyama, K. Mishima, T. Imato, T. Masadome, Y. Asano, Y. Kurokawa, H. Tabei and S. Okutani, Talanta, 58 (2002) 1123–1130. 86 N. Soh, H. Nishiyama, Y. Asano, T. Imato, T. Masadome and Y. Kurokawa, Talanta, 64 (2004) 1160–1168. 87 K. Hirakawa, M. Katayama, N. Soh, K. Nakano and T. Imato, Anal. Sci., 22 (2006) 81–86. 88 K. Hirakawa, M. Katayama, N. Soh, H. Ohura, S. Yamasaki and T. Imato, Electroanalysis, 18 (2006) 1297–1305. 89 M.A. Nelson, W.S. Reiter and D.S. Hage, Biomed. Chromatogr., 17 (2003) 188–200.

590

Jianhua Wang and Xuwei Chen

90 K. Hartwell, B. Srisawang, P. Kongtawelert, J. Jakmunee and K. Grudpan, Talanta, 66 (2005) 521–527. 91 S.M. Varnum, M.G. Warner, B. Dockendorff, N.C. Anheier Jr, J.L. Lou, J.D. Marks, L.A. Smith, M.J. Feldhaus, J.W. Grate and C.J. Bruckner-Lea, Anal. Chim. Acta, 570 (2006) 137–143. 92 H.L. Zhu, H.W. Chen and Z.L. Zhou, Chinese J. Anal. Chem., 32 (2004) 841–846. ˚ . Lernmark and J. Ruzicka, Analyst, 128 (2003) 1157–1162. 93 A.D. Carroll, L. Scampavia, D. Luo, A 94 P.L. Urban, D.M. Goodall and N.C. Bruce, Biotechnol. Adv., 24 (2006) 42–57. 95 M. Yaqoob and A. Nabi, Talanta, 55 (2001) 1181–1186. 96 J.J.J. Ruiz-Diaz, A.A.J. Torriero, E. Salinas, E.J. Marchevsky, M.I. Sanz and J. Raba, Talanta, 68 (2006) 1343–1352. 97 D. Janasek and U. Spohn, Sensor. Actuat. B., 74 (2001) 163–167. 98 R.F. Dutra, K.A. Moreira, M.I.P. Oliveira, A.N. Araujo, M.C.B.S. Montenegro, J.L.L. Filho and V.L. Silva, Electrophoresis, 17 (2005) 701–705. 99 J.Y. Wang and Z.L. Fang, Chem. Res. Chinese U., 22 (2006) 287–291. 100 J.Y. Wang, Z.L. Fang and S.K. Xu, Chinese J. Anal. Chem., 30 (2002) 307–311. 101 C.K. Pires, P.B. Martelli, B.F. Reis, J.L.F.C. Lima and M.L.M.F.S. Saraiva, J. Autom. Method. Manag., 25 (2003) 109–114. 102 P.C. Chen, B.C. Hsieh, R.L.C. Chen, T.Y. Wang, H.Y. Hsiao and T.J. Cheng, Bioelectrochemistry, 68 (2006) 72–80. 103 Z.R. Xu and Z.L. Fang, Anal. Chim. Acta, 507 (2004) 129–135. 104 N. Piza, M. Miro, J.M. Estela and V. Cerda, Anal. Chem., 76 (2004) 773–780. 105 C. Wu, L. Scampavia, J. Ruzicka and B. Zamost, Analyst, 126 (2001) 291–297. 106 Y. Chen and J. Ruzicka, Analyst, 129 (2004) 597–601. 107 Y. Chen, A.D. Carroll, L. Scampavia and J. Ruzicka, Anal. Sci., 22 (2006) 9–14. 108 N. Ohgami, S. Upadhyay, A. Kabata, K. Morimoto, H. Kusakabe and H. Suzuki, Biosens. Bioelectron., 22 (2007) 1330–1336. 109 J. Ruzicka, P.J. Baxter, O. Thastrup and K. Scudder, Analyst, 121 (1996) 945–950. 110 I. Lahdesmaki and J. Ruzicka, Fresenius J. Anal. Chem., 362 (1998) 67–72. 111 P.S. Hodder and J. Ruzicka, Anal. Chem., 71 (1999) 1160–1166. 112 P.S. Hodder, C. Beeson and J. Ruzicka, Anal. Chem., 72 (2000) 3109–3115. 113 C.M. Schulz and J. Ruzicka, Analyst, 127 (2002) 1293–1298. 114 C.M. Schulz, L. Scampavia and J. Ruzick, Analyst, 127 (2002) 1583–1588. 115 H.A. Erxleben, M.K. Manion, D.M. Hockenbery, L. Scampavia and J. Ruzicka, Analyst, 129 (2004) 205–212.