Nanosized labels for rapid immunotests

Accepted Manuscript Nanosized labels for rapid immunotests Irina Yu. Goryacheva, Pieterjan Lenain, Sarah De Saeger PII: DOI: Reference:

S0165-9936(13)00059-9 http://dx.doi.org/10.1016/j.trac.2013.01.013 TRAC 14047

To appear in:

Trends in Analytical Chemistry

Please cite this article as: I.Y. Goryacheva, P. Lenain, S. De Saeger, Nanosized labels for rapid immunotests, Trends in Analytical Chemistry (2013), doi: http://dx.doi.org/10.1016/j.trac.2013.01.013

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Nanosized labels for rapid immunotests Irina Yu. Goryacheva, Pieterjan Lenain, Sarah De Saeger This review summarizes developments in labels for rapid immunotests. Their application in bio-imaging in cellular and molecular biology, biosensing and biotechnology is becoming interesting as a tool for rapid tests and point-of-care techniques. For each label, we discuss the possibility of simultaneous detection of multiple analytes and reader application. Keywords: Bio-imaging; Biosensing; Immunoassay; Immunotest; Multi-analyte assay; Nanoparticle (NP); Nanosized label; Quantum dot; Rapid test; Visual detection

Irina Yu. Goryacheva* Saratov State University, Chemistry Institute, Department of General and Inorganic Chemistry, Astrakhanskaya 83, 410012 Saratov, Russia Pieterjan Lenain , Sarah De Saeger Ghent University, Faculty of Pharmaceutical Sciences, Laboratory of Food Analysis, Harelbekestraat 72, 9000 Ghent, Belgium

*Corresponding author. Tel.: +79 272 291 006; E-mail: [email protected]

1.

Introduction

The main tendency in the development of each new label for rapid methods could be outlined as follows: initially they are investigated as biolabels in biochemical research, followed by application in traditional immunoassay formats (“new-label linked immunosorbent assay”), and finally in the development of sensors and rapid tests. Another common tendency is a shift from the clinical area [point-of-care (POC) testing] toward the food and feed control area and finally to the field of environmental control. The immunoassay format changes in parallel: from a sandwich format for high-weight analytes of clinical interest to a competitive format for mostly low-weight analytes in ecological monitoring. Only a few publications concerning rapid tests for ecotoxicological items could be found {e.g., the detection of microcystin in surface, drinking and salt water [1]; 2,4,6-trinitrotoluene in soils [2]; and, carbofuran and triazophos in water samples [3]}. In spite of substantial interest in in-field assay, the main problem restricting application of rapid immunotests is their lack of sensitivity. Two main approaches are used to solve this: (1)improvement in the recognition system and assay optimization; and, (2)development of new labels or label systems, thereby improving analytical signal generation through higher brightness and/or reader optimization. Several reviews have been published in related areas. Data about lateral-flow immunoassay (LFIA) development were summarized in reviews of Posthuma-Trumpie et al. [4] and Warsinke [5], devoted to its application in the clinical area. The emphasis was put on the strengths and the weaknesses of this format with traditional colloidal gold nanoparticles (AuNPs) as labels and only several sentences mentioned the perspectives of applying new labels. Application of NP labels in immunosensing with optical detection was reviewed by Seydack [6]. A detailed analysis of the advantages and the disadvantages of each one was described within the 1   

framework of multiplexing, better sensitivity and stability. Progress in the development of different types of inorganic NPs used as labels in immunoassay was recently reviewed by Chafer-Pericas et al. [7] and focused on the NP-preparation methods and surfacefunctionalization procedures in order to improve the sensitivity of the immunoassays involved. A review concerning the trends in fluorescence immunochromatography was published by Pyo and Yoo [8]. Here, we present an overview of the nanosized labels for rapid immunochemical tests, the principles of signal generation and the features of applications. Focus lies on LFIA as it is the most occurring rapid test format, as well as the most promising technique from our perspective. Optimal labels should be colloidal in water, have uniform size and shape, be easily conjugatable to biomolecules, be able to produce an intense analytical signal and show stability against aggregation during the test-preparation and assay procedures. Unlike other kinds of label, NPs have common properties (e.g., photostability, long shelf life and size-dependent mobility). Table 1 gives an overview of advantages and disadvantages of the labels involved. Usually, labels with sizes of 15–800 nm allow unobstructed flow through the LFIA membranes [4].

2. Colored nanoparticles 2.1. Colloidal gold Colloidal gold is the most popular label for rapid tests and it is also often referred to as the most stable of all colloids [6]. The first application of AuNPs conjugated with antibodies in an immunoassay was reported in 1981 [9]. AuNPs have unique physical properties that depend on their size, shape and the inter-particle distance. One important advantage of gold labeling over competing methods is that there is no known toxicity. This simplifies biological application of this label by removing the necessity of covering the toxic core with inert material. The functionalization of AuNPs could be achieved by linking various bio-functional groups (e.g., amphiphilic polymers, silanols, sugars, nucleic acids and proteins) via the strong affinity of the gold surface with thiol ligands [9]. AuNPs are little susceptible to aggregation during the preparation of the test devices. The colors of AuNPs, the basis for their application as markers, are due to the presence of a surface-plasmon-resonance (SPR) (or localized SPR) absorption band, which occurs when an incident photon frequency is in resonance with the collective excitation of the conductive electrons of the particle [6]. The color of AuNPs and their corresponding wavelength of maximal absorbance strongly depend on the size and the shape of the AuNPs. The brightness of AuNPs depends on their size, which can be controlled easily during manufacturing. The sensitivity of 30-nm particles is 2–4 times better than that of 15- standard fluorescent labels (i.e. Cy-3 and Cy5). However, according to Khlebtsov and Khlebtsov [12], the experimental work with particles exceeding 30–40 nm is difficult because of colloid-stability problems. Colloidal gold conjugation has become a standard method in immunochromatographic tests in very different areas (i.e. POC, then food control and recently environmental control). One of the most popular AuNP-based formats is LFIA. It has wide application in the areas of POC, but less for food control and environmental purposes. Spread of the colloidal gold-based LFIA is hampered by the low sensitivity of the label. An attempt to use AuNP-based LFIA for lead-ion detection in water samples has been published [13]. Also DNA-based and aptamerbased lateral-flow dipsticks with AuNP labels have been reported for detection of DNA [13], adenosine [14] and lead [15]. Readers for AuNP-based LFIA are commercially available. Application of AuNP in multi-analyte assay is only possible using separate test-zones. By use of a galvanic replacement reaction between the Ag atoms of silver nanocubes and Au ions of tetrachloroauric acid, particles of different color were obtained. Depending on the Ag/Au conversion ratio, the particle-plasmon resonance was tuned from 450 nm to 700 nm, and the suspension color changed from yellow to blue. The multiplex capability of yellow, red and blue particles was illustrated in a dot-immunoassay format [16]. 2   

Several approaches to signal enhancement have been developed and the two main ones are: (1) modification of AuNPs without complicating the assay procedure further; and, (2) assay sensitivity is improved by incorporating additional steps to the procedure. The signal enhancement step is usually realized after the standard assay procedure with AuNPs, so LFIA becomes a multiple-step format in this way. 2.1.1. Modification of gold nanoparticles. Modification of AuNPs allows sensitivity enhancement without additional assay steps. Core-shell NPs are becoming increasingly popular because of their brightness. Khlebtsov and Khlebtsov [12] performed theoretical estimates and showed that the dot extinction of 100-nm silica/gold nanoshells can be 1000 times greater than that for the same number of 15-nm AuNPs. Liao and Li [17] published an immuno-dipstick with core-shell silver-AuNPs, which showed greater intensities than pure AuNPs. These authors also mentioned the application of core-shell nanocomposites, which result in a strong enhancement of aflatoxin B1 sensitivity while possessing reproducibility and stability comparable to traditional AuNPs. Application of a detector reagent consisting of nano-Fe2O3 particles as core and AuNPs as a shell for LFIA detection of aflatoxin B2 allowed Tang et al. [18] to increase sensitivity three-fold compared to conventional AuNPs. 2.1.2. Signal enhancement procedures for gold nanoparticles. 2.1.2.1. Silver enhancement. Upon addition of silver-containing enhancement buffer, metallic silver deposits on AuNPs cause obscuration, which can be measured optically by a CCD camera, scanner or visually. The chemical basis for silver intensification via colloidal gold, known as autometallography, dates back to 1930 and its application as a gold tracer linked to an antigen– antibody reaction was first demonstrated by Scopsi et al. [19]. Gold provides a route for the transfer of electrons from the reducing agent in solution to silver ions bound to the gold surface. This results in specific deposition of metallic silver at the site of gold labeling, and the silver can then participate again in the catalytic reaction. The sensitivity of immunogold detection can be increased approximately 100-fold by silver enhancement [20]. This approach is restricted by the need for additional steps and the very strong requirements for preceding washing steps to remove interfering ions (e.g., chlorine). Some applications of silver enhancement in different formats have been published. A multi-well chip was developed for human immunoglobulin G detection using a CCD camera [21]. Wu et al. [22] used polydimethylsiloxane-AuNP composite film as the basis with silver enhancement for colorimetric detection of cardiac troponin I. The obscuration due to silver deposition was relative to the amount of antigen because of the difference in inhibiting ability between silver enhancement blocked with bovine serum albumin (BSA) film on the one hand and antibody-antigen complex on the other. Cross-flow chromatographic analysis was recently described [23] as a variant of the LFIA procedure. It is interesting to mention that silver enhancement could be applied for not only AuNPs, but also increasing sensitivity of silver-NP-based LFIA [24]. 2.1.2.2. Gold-nanoparticle enhancement. In the group of “sensitizers”, application of AuNPs itself as an enhancement reagent could be singled out. After performing the normal method, the AuNPs conjugated with primary antibody will be captured by secondary antibodies on the test line of the strip [25]. As a result, AuNPs accumulate at the test line and the sensitivity is greater. Nagatani et al. [25] reported a 50-fold decrease in limit of detection (LOD) using this procedure (25 pg/mL and 1.0 ng/mL of human chorionic gonadotropin hormone with and without “signal enhancement” procedure). However, a variant of this approach without any additional steps also includes two types of bio-conjugated AuNPs encapsulated in different pads – one type being conjugated with antibody and blocked with BSA, and another with anti-BSA antibody. Choi et al. [26] found that the size of the two conjugates is very critical for the LOD. 3   

When 10-nm AuNPs were used conjugate and 40-nm AuNPs for the second, the sensitivity increased about 100-fold compared to conventional LFIA. An additional advantage of the AuNPenhancement procedure is that the same reader can be applied as for normal AuNP-based method. 2.1.2.3. Enzyme enhancement. Application of AuNPs as a label enhanced with horseradish peroxidase (HRP) improves optical detection. The strips could be read with the strip reader to obtain the calibration curve corresponding to the AuNPs used as “direct” labels. After the first reading step, the LFIA strips could be dipped into the HRP substrate, washed and read again.  After application of chromogenic substrate, a darker color appeared with enhanced intensity compared to the red color of the unmodified AuNPs. Parolo et al. [27] compared three different substrates and found an increase in sensitivity up to an order of magnitude. 2.1.3. Catalytic properties. The use of the catalytic properties of AuNPs allows their application in chemiluminescent detection, as was shown in a microplate format based on luminol–AgNO3– AuNPs. AuNPs could trigger the reaction between luminol and AgNO3, accompanied by light emission. This approach has been used for human immunoglobulin G detection. Compared with other reported chemiluminescent systems, the proposed protocol avoids a strict stripping procedure or synthesis processes difficult to control, making the method simpler, time saving, easily automated [28] and applicable in rapid test format. 2.1.4. Distance-dependent properties. Another principal way for AuNP assays with visual detection based on their distance-dependent properties was introduced for the first time by Elghanian et al. [29] for polynucleotide detection based on the color change of NPs. When individual AuNPs come into close proximity (the center-to-center distance is usually smaller than 2.5 times the diameter of the AuNP), their individual surface plasmons combine (so-called inter-particle surface-plasmon coupling), which results in a change of color from red to purple and blue. A similar principle was used in homogeneous immunoassay, using human serum albumin as a model analyte. Binding of antibodies to functionalized NPs causes a shift of the visible absorption maximum of AuNPs. Quantification of the analyte could be obtained after competitive inhibition [30]. The distance-dependent properties were also used for rapid colorimetric detection of small analytes (e.g., melamine, dopamine and ascorbic acid). Because of the presence of three aminogroups, melamine could rapidly induce aggregation of label-free AuNPs, resulting in a red-toblue (or purple) color change. Li et al. [31] showed that the concentration of melamine in raw milk can be determined by monitoring with the naked eye or a UV-Vis spectrometer with an LOD of 0.4 mg/L. The method is rather simple and the whole process takes only 12 min, including sample pretreatment (centrifugation with trichloroacetic acid, pH adjusting and filtering). Guo et al. [32] proposed a similar method for milk and infant formula with an LOD of 1.0 mg/L and 4.2 mg/L, respectively by naked eye, and 0.15 mg/L and 2.5 mg/l, respectively, with UV-Vis-spectroscopy within 30 min (sample preparation includes ultrasonification with trichloroacetic acid and chloroform, centrifugation and pH adjustment). Comparison of several modifiers showed that the modification with cystamine, aimed at weakening the electrostatic repulsion force between the AuNPs, increased the signal intensity about a 100-fold compared to the unmodified AuNPs [33]. A similar approach was used for the detection of dopamine; the presence of Cu2+ ions increased sensitivity through complexation of two dopamine molecules [34]. Ascorbic acid could also induce a rapid aggregation of azidefunctionalized and alkyne-functionalized AuNPs in the presence of Cu2+, thereby resulting in a color change [35]. This distance-dependent approach was also realized in a solid-based application, which is more suitable for practical use. Liu and co-workers [14] first developed a lateral-flow device 4   

where AuNPs aggregate upon addition of a specific target analyte, after which they dissociate into well-dispersed AuNPs. These dispersed AuNPs are modified by a biotin flow along a cellulose-based membrane and are captured by a coated line of streptavidin on the membrane. A red color appearing on the streptavidin line indicates the presence of the target analyte. Inspired by this work, Zhao et al. [36] have demonstrated the feasibility of using an AuNP-based dotsensing platform on paper strips. DNA-cross-linked AuNP aggregates were spotted on paper, and a red color signal was initiated in the presence of the target DNA by re-dispersion of the AuNP aggregates on paper. New sensor formats, based on colorimetric detection, are reviewed by De la Escosura-Muсiz et al. [37]. 2.2. Colloidal carbon The black color of carbon NPs (CNPs) can be visually detected with high sensitivity and was first introduced as a label for rapid immunochemical testing in 1993 by van Amerongen et al. [38]. Preparation, functionalization and application of such CNPs for rapid immunotests were recently reviewed by Posthuma-Trumpie et al. [39]. CNPs are very inexpensive, available in large batches and very suitable for quantification of results using “gray pixel” processing because of their “black-on-white” test results. Due to the high extinction of carbon black, the detectable concentrations of the pure particles are 0.04 ng/mm2 (0.02 amol/mm2) using a scanner and 0.2 ng/mm2 (0.1 amol/mm2) by the naked eye [40]. Lonnberg and Carlsson [40] compared the detection ability to those obtained with enzymatic labels: alkaline phosphatase (AP) using a substrate yielded a chemiluminescent signal (0.02 amol AP/well); the use of -galactosidase and a substrate resulted in a fluorescent signal (0.3 amol βgalactosidase/well); and, HRP using a substrate rendering a colored signal (5 amol HRP/microtiter well). The sensitivity of LFIA with carbon-black labels was reported to be equal to that of the corresponding ELISA [41]. It was also shown that carbon black had a remarkably low LOD of 0.01 μg/mL compared to 0.1 μg/mL, 1 μg/mL and 1000 µg/mL for silver-coated AuNPs, AuNPs and polystyrene beads, respectively [42]. High sensitivity compared to other LFIAs and even sensitivity 50–100 times more than corresponding enzyme-based immunoassays have been achieved by utilizing large carbon-black nano strings (this dimension corresponds to a spherical particle of about 200-nm diameter) in combination with sensitive image-scanner detection [43]. 2.3. Colloidal selenium Rust-colored colloidal selenium ( max = 540 nm) was employed for detection of lipoprotein A in plasma by use of LFIA with visual detection. The authors used several test lines and evaluated the analyte concentration based on the color intensity of these test lines in a semi-quantitative assay. Lipoprotein A coated colloidal selenium was stable from two months in liquid form and up to over eight months in lyophilized form, and tests showed reproducible results during a fivemonth period [44]. Unfortunately, the authors had not compared sensitivity, stability and robustness of the test based on colloidal selenium labels to tests using other labels. Therefore, it is difficult to draw conclusions about the prospects for this label. Since this first publication in 1993, there has been no further application of colloidal selenium in rapid tests. 2.4. Colloidal iron oxide Fe3O4-NPs are applied as labels mostly because of their magnetic properties (Section 4). However, their optical properties could be helpful when they are used as colored labels. The disadvantages of Fe3O4-NPs in comparison with AuNPs are: (1) the absorption spectrum of Fe3O4-NPs is wide, covering almost the whole visible range due to the intraband transition; 5   

(2) the dark brown color of Fe3O4-NPs is apparently not as bright as that of the AuNPs. Nevertheless, the integral molar absorption coefficient of Fe3O4 nanocrystals within the visible light range is rather comparable with that of the AuNPs. Furthermore, aggregation hardly changes the absorption properties of Fe3O4-NPs, where it does change those of AuNPs [45,46]. Fe3O4-NPs were used as colored labels for LFIA in the detection of the pesticide paraoxon methyl, obtaining a LOD of 70 ng/mL. More interestingly, application of Fe3O4-NPs aggregates demonstrated that the LOD could be decreased over 40 times, reaching 1.7 ng/mL. Visual LODs were 1000 ng/mL and 150 ng/mL for NPs and aggregates as labels, respectively. The Fe3O4-NPs aggregates were prepared by crosslinking Fe3O4-NP-bearing surface carbonyl groups with poly-L-lysine [45]. An additional advantage of this label is the possibility to use two analytical signals (optical and / or magnetic) with one test without additional steps. 2.5. “Colloidal” dyes Single-dye molecules do not possess enough chromophore color intensity, so “multi-loaded” labels were developed. Only a few applications of colloidal dyes as labels for clinical assay were developed. Blue colloidal dye (D-1) and disperse dye (Dadisperse navy blue SP) were used as labels for immunochromatographic strips [47], red colloidal dye (R-3) was used for a flow-through assay and the results were similar to those detected by routine ELISA [48]. It was shown that these kinds of label are suitable for both quantitative and qualitative detection. Another approach to enhance the chromophore color intensity of dye molecules is to load them to polylysine of different molecular weight. The optimized dye chromophore using polylysine with a molecular weight of 189.4 kDa and a molar ratio (mol dye/mol amine group in polylysine) of 1.5 was used for labeling a model antibody in LFIA. Loading of polylysine with reactive dyes of different colors allowed [49] multiple analyte detection in a single qualitative or quantitative (by using the table-top densitometer) immunochromatographic assay. A new principle of label creation is by application of NPs containing a precursor of highlycolored compounds. This type of label, which is based on organic NPs, is loaded with the colorless indigo precursor 5-bromo-4-chloro-3-indolyl acetate and coupled with antibody for LFIA detection of a model protein [50]. Through hydrolysis, this precursor produces a bluecolored compound, 5-bromo-4-chloro-3-hydroxyindole, which forms an insoluble blue-color precipitate after oxidation, 5,5’-dibromo-4,4’-dichloro-indigo. The hydrolysis was performed by adding a developing reagent composed of 2-propanol, NaOH and H2O2, which transforms the precursor into the blue-colored precipitate. Comparison of the proposed label with traditional AuNP labeling demonstrated better assay-performance characteristics of the proposed label (two times higher signal-to-noise ratio).

3. Luminescent molecules and nanoparticles Reporter systems with luminescent detection are now widely used in research and clinical diagnostics because they provide high sensitivity and are capable of simultaneous use of multiple labels with different spectral characteristics (multiplexing). In some cases, the sensitivity of luminescent labels is comparable with that of enzymatic labels, but the assay procedure is essentially simpler. As the chemical nature of luminescent labels with similar optical properties can be very different, they are classified in this section according to their principle of luminescence. In general, emitters can be categorized as down-converting and up-converting. Downconverting emitters absorb a photon and then emit a photon with lower energy. This is the more common process and it is exhibited by fluorescence, phosphorescence and most of the betweenmolecules energy-transfer processes. By contrast, up-converting phosphor absorbs two or more photons with an energy that is lower than the energy of the emitted photon. 6   

3.1. Fluorescent dyes The simplest kind of luminescence emitters are single-molecule fluorescent organic dyes. Historically, fluorophores (e.g., fluorescein, rhodamines and cyanine dyes) have been used as cell and tissue labels in fluorescence microscopy and cell biology [51].  Now, several series of fluorescent dyes are available with improved fluorescent characteristics (high quantum yield, relatively large Stokes shift, chemical and photo stability) (e.g., Alexa Fluor from Invitrogen and Molecular Probes, PromoFlor from PromoKine, DyLight Fluor from Dyomics, ATTO Dyes from ATTO-TEC, Hilyte Fluor from AnaSpec). These dyes have potential to be applied as fluorescent labels for rapid tests. It is important to mention that the application of fluorescent labels to membrane tests is complicated by high light scattering caused by the membrane support. There is also interference of internal fluorescence, originating from proteins (antibody), probe components, and analytes (e.g., polycyclic aromatic hydrocarbons and some mycotoxins). Weaknesses of fluorescent dyes, compared to the NPs, are low photostability, high quenching by the environment and quenching of concentration [51]. To increase the brightness of the labels, multiple fluorescent dyes can be coupled to a carrier and subsequently to immunoreagents. This means a high label-to-antibody ratio and, as a consequence, the signal intensity is greater than standard conjugation of antibody to label. Another way to enhance fluorescence intensity is use of particles (e.g., polystyrene) loaded with organic fluorescent dyes. Various such microspheres are commercially available (e.g., FluoSpheres from Invitrogen). Similarly, fluorescent dye-doped silica NPs can be applied [52]. These approaches make labels brighter but do not solve problems with broad emission and small Stokes shift, thus resulting in cross-talk between excitation and emission signals. Kim et al. [1] examined several fluorescent dyes, including fluorescein isothiocyanate, rhodamine, Texas Red, Alexa Fluor 488 and Alexa Fluor 647, and reported Alexa Fluor 647 to be more stable and to give a bigger fluorescent signal than the others. Alexa Fluor 647 was reported in a highly-sensitive LFIA for the detection of prostate-specific antigen [53], C-reactive protein [54] and human-serum albumin [55] in whole blood without interference from blood compounds. For POC testing, it is important to apply the test directly to whole blood, because the application of the test to plasma and serum requires blood pretreatment. A similar test was also developed for albumin detection in urine [56]. There was good correlation between the results of these tests and those comprising more complicated and timeconsuming methods. In environmental assays, a fluorescent dye-based LFIA was developed for the quantification of microcystins in surface water [1]. Sensitivity and reproducibility of immobilized-antibody and immobilized-antigen systems were compared for these low molecular weight analytes and the superiority of the immobilized-antigen system was shown. Application of home-made [1] or commercial (i-CHROMA, BioditechMed, Korea) laser fluorescent scanners obtained quantitative results. Cibitest’s FLORIDA device consists only of an excitation lamp, and reading can be performed by the naked eye. Embedded Systems Engineering (Germany) created a miniature confocal optical sensor that can be modified with a wide variety of fluorescent labels for evaluation of quantitative results. 3.2. Quantum dots Another type of label useful for visual and instrumental detection of rapid-test outcomes is the quantum dot (QD). QDs are inorganic luminescent semiconductor nanocrystals, exhibiting sizedependent luminescence emission spectra. In recent years, QD applications have become the most extensive area of immunoassay labeling. Various types of QDs (e.g., InP, InAs, GaAs, GaN, ZnS and ZnSe) have been synthesized for different research applications, as have QDs comprising heavier atoms (e.g., CdTe, HgSe or 7   

PbSe). However, the most popular core for bioassay application is CdSe because different-sized QDs emit light across the whole visible spectrum. Usually, the CdSe-core radii vary in the range 1–6 nm, which is known to be smaller than the bulk exciton Bohr radius (which, for CdSe, equals 6 nm). When the radius of the QDs is smaller than the bulk exciton Bohr radius, it strongly influences the energy bands under quantum confinement. Combined with a decrease in QD radius, this causes a blue shift in the emission spectrum. During fabrication, the diameter of QDs can be selected to achieve emission of a variety of colors. However, uncovered CdSe nanocrystals have insufficiently high quantum yields and stability in aqueous solutions due to surface-related trap states acting as fast non-radiative de-exciting channels for photo-generated charge carriers [57]. Coating of core nanocrystallites with higher band-gap inorganic materials (shell) has been shown to improve the photoluminescent quantum yields by passivation of the surface non-radiative recombination sites. Several wide band-gap semiconductors (ZnS, CdS or ZnSe) can be used as shell material. ZnS is the most popular shell material, due to its wider band gap (energy difference between the valence band and the conduction band), thus allowing the efficient confinement of both the photo-generated electrons and holes in the nanocrystal core. An additional middle shell (CdS or ZnSe) located between the CdSe core and the ZnS outer shell could be added to reduce the internal strain of the nanocrystals caused by CdS and ZnSe intermediate-lattice parameters compared to those of CdSe and ZnS. QD-extinction coefficients are ~105–106/M/cm, depending on the particle size and the excitation wavelength, which is about an order of magnitude higher than organic dyes. The quantum yields of CdSe QDs vary up to 40%. In comparison to classical organic dyes (e.g., rhodamine 6G and fluorescein), CdSe nanocrystals show lower quantum yields at room temperature, but the lower quantum yields are compensated for by their larger absorption crosssections. Chan and Nie [58] estimated that each CdSe/ZnS QD is about 20 times brighter and 100–200 times more stable than rhodamine 6G. Opposite to organic fluorescent dyes, QDs have symmetric, narrow-emission bands and their absorption spectrum reaches into the UV area regardless of their size. Their spectral characteristics therefore allow simultaneous excitation of particles with different sizes using a single wavelength, resulting in emission at multiple wavelengths. These characteristics make them ideal labels for detecting multiple binding events in one spot. Weak points of QDs (e.g., toxicity, water insolubility and absence of functional groups available for bioconjugation) were overcome by the introducing QDs into silica nanoshells [59]. Another way to achieve water solubility and biocompatibility is conjugation with a bi-functional molecule (e.g., mercaptoacetic acid) [58]. QDs have been widely applied for biolabeling in the fields of cell biology, molecular biology, genomics and medical diagnosis, and are commercially available in free and conjugated forms (Invitrogen, and Evident Technologies). Water-soluble CdSe-ZnS NPs, compatible with conjugation methods in aqueous conditions, are usually prepared through a stepwise procedure, comprising core CdSe-nanocrystal growth, ZnS coating, capping with an organic layer and finally size-selection precipitation [60]. Broad excitation and narrow emission bands simplify the use of QDs for multi-analyte assays and minimize matrix influence. This latter aspect is especially important for blood-sample analysis. Also, the luminescent lifetime of QDs tends to be in the range 30–100 ns, which is slightly longer than those of organic dyes (1–5 ns), but much shorter than those of lanthanide probes (1 µs–1 ms). These excited-state decay rates last much longer than the background fluorescence and Raman scattering of most sample matrices. One can therefore use time-gated detection to reduce or to remove background fluorescence selectively. CdSe-ZnS core-shell QDs were used as luminescent labels for a microtiter-plate assay (fluorescence-linked immunosorbent assay or FLISA) toward the detection of various analytes in different matrices. Comparison of the analytical parameters of ELISA and FLISA showed a fourfold decrease in IC50 (0.4 ng/mL and 0.1 ng/mL for zearalenone detection with ELISA and FLISA, respectively) [61]. A specific name for QD-based immunoassay was suggested – QLISA [62]. 8   

QD luminescent properties were successfully combined with enzyme chemiluminescent labels for simultaneous detection of three cancer markers in human serum [63]. QDs were applied as a label for LFIA, which was patented for detection and quantification of one or more analytes [64,65]. A review on the Web of Science indicates that no manuscripts were published on LFIA with QDs as a label prior to 2009, and only some preliminary results were reported. QDs as labels for LFIA were described for small molecule trichloropyridinol [66], for protein biomarker nitrated ceruloplasmin [67] and syphilis antigen [68]. Comparison of LFIA sensitivity with the same set of immunoreagents showed a 10-fold decrease in the LOD by naked-eye CdTe QD test strips (under a portable UV lamp) compared to colloidal gold test strips [68]. Similarly, the sensitivity of column gel-based immunotests for benzo[a]pyrene detection in water showed a lower LOD of 5 ng/L using HRP or CdSe QDs as a label, but 25 ng/L in the case of AuNPs. Assays with particle labels required only four consecutive working steps, whereas those based on HRP required five additions of reagent [69]. Column QD-based immunotests with polyethylene frits and sepharose gel were used for zearalenone detection in wheat [61]. With all the above-mentioned advantages and perspectives, we can expect more rapid tests using QDs soon. 3.3. Infrared emitters Infrared (IR) emitters are based on the near-IR (NIR) emission of compounds containing Nd3+, Er3+ or Yb3+ ions and are less studied and used than Eu3+ and Tb3+. This lack of research can be ascribed mainly to the technological advances in new detectors and excitation sources, which have been developed in the past decade [70]. An Nd3+-doped Y2O3 matrix can absorb in both visible and NIR regions (500–900 nm) with subsequent emission in the NIR region [71]. The use of functionalized Y2O3:Nd3+ NPs as a label showed some advantages based on the internal properties of Nd3+ luminescence. It succeeded in eliminating the interferences from the biological fluorescence background without using timeresolved techniques. Also, excitation in the NIR region (500–900 nm) could be easily performed with lasers and LEDs without inducing electronic excitation in the biological medium. An attempt to use such labels in biological assays was reported by Kodaira et al. [60] as heterogeneous immunoassay for oxidized lipoprotein detection. While an application of this label was reported for only microplate format, it seems a very promising label for rapid tests if a suitable reader would be available. 3.4. Up-converting emitters Up-converting phosphors (UCPs) utilize a combination of absorber and emitter ions in submicron-sized crystals (~200–400 nm in diameter). The absorber ion (energy donor) is excited by the energy of an IR light source, usually a 980-nm diode laser. This energy is transferred nonradiatively to the emitter ion (energy acceptor) which radiates a photon in the visible – NIR range (400–800 nm) depending on the ion composition of the crystal. This anti-Stokes’ photoluminescence is based on the sequential absorption of two low energy photons. In contrast to conventional two-photon excitation, the simultaneous absorption of photons is not necessary for up-converting processes. It can occur in the microsecond time scale because of the long lifetime of the metastable excited states. This enables the excitation of UCP by low-energy IR photons, produced by a laser with relatively low power. In such conditions, the photodegradation of biomolecules and the luminescent background are not crucial factors. This technology is also compatible with optically-difficult samples (e.g., whole blood). Because up-conversion processes are unique with regard to the lack of background luminescence from the biochemical assay, there is no need for time-resolved detection. The emission wavelengths differ distinctly from the excitation wavelength in the anti-Stokes area, so the detection process can be simplified and made robust with respect to environmental sampling conditions [72,73]. 9   

Inorganic materials capable of up-conversion consist of a host lattice embedded with certain rare-earth dopant ions. So far, the most efficient up-converting materials reported have been based on hexagonal sodium-yttrium-fluoride (NaYF4) host lattices. The dopant ions in the UCPs are typically trivalent lanthanides, which possess multiple, long-lifetime excited states [74]. Different combinations of rare-earth emitting (erbium, holmium, thulium) and absorbing (ytterbium, erbium, samarium) ions have created more than 20 unique UCP compositions [75]. The optical properties of the UCP particles are completely unaffected by their environment, since the energy-transformation process occurs within the host crystal [110]. In addition, UCPs have a narrow emission band, long fluorescent lifetime, high chemical stability, and low potential cytotoxicity [76]. A simple way to obtain sub-micron-sized UCP particles is by bead-milling commercially available bulk phosphor material (Orasure Technologies, Inc.; Phosphor Technology Ltd.). The smaller particles are subsequently fractionated by sedimentation or flow filtration, based on multiple filter membranes with decreasing pore sizes. However, irregular shapes still remain a problem, especially in heterogeneous bioanalytical assays. Large distribution of sizes and nonspecific binding of UCP particles can cause a large assay variation in the analyte’s lower concentration range and thus restrict the analytical LOD. To improve homogeneity in both size and shape of UCP particles, the homogeneous precipitation method, followed by a series of fluidized-bed processes, has been used. In past years, several new methods of synthesis were reported [74]. A disadvantage of UCP technology is the relatively low quantum efficiency of the upconversion process. Also, the sub-micron size of UCP particles is quite large relative to the typical protein size and can affect the specificity and the kinetics of assays because of steric restrictions on the bead surface. Phosphors with diameters of 200 nm or less address particle-size issues to some degree [75]. Another disadvantage when using UCP particles as labels is the nonspecific binding of untreated phosphor particles with biomolecules [73]. Similar to QDs, UCP particles cannot readily conjugate with immunoreagents. Surface functionalization of these particles not only broadens bio-chemical compatibility and minimizes non-specific binding, but also increases the stability of UCP particles by enhancing the aqueous dispersability and reducing the tendency to aggregate [74]. Commonly, the phosphor surface is coated with tetraethylorthosilicate to obtain a thin (5–50 nm) surface layer of silica. Silanization introduces functional groups to the surface of the particles, which serve as binding sites for biomolecules (e.g., antibodies, antigens, biotin, and others) by using standard cross-linking chemistry. As a result of the common silica-coating process, the biomolecule-coupling chemistry is identical for different phosphor crystals [72]. An alternative way for bioconjugating UCP particles is passive coating with poly(acrylic acids) to attach carboxylic-acid groups to the surface of phosphor particles, followed by activation of carboxylated phosphor and coupling of biomolecules [73]. Various UCP-LFIA strips have been developed for the POC testing, combining UCP as a reporter with the LFIA principle and small, robust optical systems offering accurate quantitative detection. Tests for high-molecular-weight analytes based on a sandwich format were reported for Schistosoma circulating anodic antigen [77], Escherichia coli [78], Yersinia pestis [79], respiratory syncytial virus [80], interferon γ [81], pathogens Streptococcus pneumonia [82] and Brucella [83], hepatitis B surface antibody [84] and nucleic acids [85]. Simultaneous detection of two biomarkers in blood samples was realized with LFIA for the diagnosis of mycobacterial diseases and showed good correlation with ELISA [86]. A competitive assay format was applied for drugs of abuse detection in saliva samples [78]. A reader called UPlink from Orasure Technologies, Inc. (Bethlehem, PA, USA) has been developed for UCP-based LFIA. The lowest detectable concentration for the UCP particles was 10–100 emitting particles, depending on their properties [78]. Also, a standard epi-fluorescence microscope was adapted to visualize antigens in tissue sections or on cell membranes through the eyepieces by using UCP particles [87]. 10   

Several investigations compared the sensitivity of UCP particles as labels with other reporter systems and the sensitivity of UCP-LFIA with other immunochemical methods. Hampl et al. [72] indicated at least a 10-fold increase in sensitivity over conventional LFIA reporter systems (e.g., colloidal gold or colored latex beads) and ELISA microtiter-plate assay. Li et al. [84] reported the best sensitivity of the UCP-LFIA, in comparison with the Abbott Axsym AUSAB assay and commercial ELISA test kits. The availability of UCPs with different compositions possessing unique narrow-band emission spectra, all which can be excited by the same light source, allows multiple assays. This is done by spotting immunoreagents not only on multiple separate zones, but also within a single zone. Application of two UCP labels for simultaneous detection of two analytes was demonstrated with mouse IgG and ovalbumin (OVA) as target molecules. The two targets were color coded using UCP crystals with different compositions: thulium-oxysulfide phosphor, which emits at 480 nm (blue), and erbium-oxysulfide phosphor, which emits at 550 nm (green) [72]. The ability of the UPlink instrument to detect up to 12 lines on a single assay strip was shown and applied in the detection of a range of narcotics (including amphetamines, methamphetamine, phenylcyclohexylpiperidine and opiates) with a single multiplexed LFIA strip [78]. Corstjens et al. [88] developed multiplex UCP-LFIA for the detection of human antibodies against human-immunodeficiency virus with additional capture zones to detect antibodies against Myobacterium tuberculosis or hepatitis C virus. The authors noted that further studies are necessary to evaluate the maximum number of test lines that can be applied to a LFIA strip with special attention to the effect of large signals in the test lines closest to the sample pad. Luminescence quenching can be mentioned as a way of applying UCPs and other luminescent materials. If a specific antibody is attached to the UCP surface and analytes compete for binding with the analyte-luminescence quenching dye conjugate, this will result in an increase of the luminescent signal of the test zone [89]. 3.5. Nanoparticles with long-lived emission In contrast to standard luminescence methods that use only optical filters to separate the emission from the background light through wavelength differences, time-resolved luminescence techniques separate the emission of interest from the background through lifetime differences. Despite a relatively low intensity compared with conventional luminescence, time-resolved luminescence detection techniques have potentially higher sensitivity because of lower background noise. Time-resolved based techniques involve exciting a long-lived emitter with a short pulse of light followed by a waiting period to allow the decay of unwanted emission to a low level, before collecting the remaining long-lived signal [90]. The drawback of this technology is that only a limited number of discovered probes possess sufficient emission lifetime to differentiate significantly from the typical background fluorescence. Time-resolved reporters usually have a decay time of several hundreds of microseconds, optimally >500 μs. This is far more than that of conventional fluorescent probes or autofluorescent samples, typically showing decay times of <50 ns. Thus, the autofluorescence of blood and serum can also be rejected by time-resolved luminescence measurements. Long emission lifetime is also critical for constructing simple, low-cost instruments for timeresolved measurements [91]. A portable time-resolved luminescence reader without expensive optical filters, essential for the conventional fluorescence detection technique, was described by Song and Knotts [90]. The most common labels for time-resolved fluorescence are based on lanthanide chelates (mostly europium) because of their high luminescence quantum yield, large Stokes shift, and, more importantly, long lifetime. Fluorescence quenching due to water molecules usually leads to weak luminescence of lanthanide chelates in aqueous solution. Incorporating these chelates into sub-micron particles is an advantageous way to obtain bright fluorescence (by multiple loading and decrease of fluorescence quenching by water molecules), chemical stability and straightforward bioconjugation possibilities. For example, up to 7x105 europium-chelate molecules could be 11   

covalently loaded on each silica NP [92]. This kind of label is commercially available (Molecular Probes Inc., Seradyn Inc.). The lowest detectable amount reported was 3.3x107 particles/mm2 [93]. Different lifetime and spectral characteristics of lanthanide chelates allow their application in the simultaneous detection of two different analytes in one physical location. Hagan and Zuchner [94] recently discussed the potential of lanthanide time-resolved luminescence to design sensitive, specific immunoassays, techniques for labeling biomolecules with lanthanide-chelate tags, microtiterplate-based assays and application in luminescence microscopy. Application of these labels was published for LFIA detection of eosinophils and neutrophils in whole blood [93] and C-reactive protein in serum [90]. An additional advantage of lanthanide chelates is their high Stokes shift (>150 nm), which enables elimination of the very high background fluorescence measured by a conventional fluorescence system. This background signal originates from the scattering caused by the membrane itself, even without application of time-resolved techniques. The stationary mode of excitation and emission registration allows quantification with digital camera or even visual evaluation using a UV source. Chen et al. [95] showed that the sensitivity of therbium-chelate-loaded NPs increased over a 100-fold by use of fluorescein-isothiocyanate molecules. Xia et al. [96] compared LFIA for hepatitis B surface antigen with different labels and reported a 100-fold higher sensitivity with europium-chelate-loaded silica NPs compared to colloidal gold-based LFIA (0.03 g/L and 3.51 g/L, respectively). Other time-resolved reporters are phosphorescent chelates. Platinum, palladium and ruthenium chelates possess lifetimes of several hundred microseconds. Ruthenium complexes excite in the blue range and emit in the red range (600–700 nm). Application of phosphorescence for time-resolved measurements demands the deoxygenation of working media because of the very effective oxygen quenching of long-lived triplet states. One way to prevent oxygen quenching is to encapsulate the phosphorescent molecules in an oxygen-low or oxygen-free matrix. For example, phosphorescent molecules have been encapsulated inside polyacrylonitrile, polystyrene and Sephadex particles. Recently, halogen-containing polymers and copolymers were found to be excellent encapsulating matrices for phosphorescent chelates to provide phosphorescent NPs with surface functional groups. The sensitivity of LFIA with two kinds of time-resolved labels (commercial europium chelate loaded NPs and synthesized phosphorescent NPs) was compared by Song and Knotts [90]. They reported that europium-based labels were capable of detecting 0.2 ng/mL Creactive protein in serum. Time-resolved phosphorescence from phosphorescent NPs incorporated in a lateral flow device was also measured by a time-resolved luminescence reader developed in-house [90].

4. Magnetic nanoparticles Magnetic NPs (MNPs) as carriers and labels have been widely used in biochemical separation and detection, blood detoxification and drug delivery [97]. Application of MNPs as labels for rapid tests eliminates some disadvantages of optical and non-instrumental visual detection. As support materials are usually not entirely transparent for light in visible and near-UV spectral areas, the signal can be collected from only the top layer of the support. For example, for a nitrocellulose membrane (the common solid support for LFIA), only the signal coming from the top 10 μm of the membrane (usually 100 μm thick) can be detected [98]. In contrast, all magnetic signals originating from the MNPs within the entire volume of the membrane can be detected using a highly sensitive magnetic-particle detector. Since none of the generated signal is lost, this improves the sensitivity of the tests. Furthermore, MNP application is associated with low background noise because extremely low magnetic signals can be detected for most of the biological samples [98]. Unlike other kinds of NP, MNPs give signals that do not degrade over 12   

time. Another common feature is that the size of the MNPs largely determines flow rate and affects detection time. The magnetite content of MNPs is positively correlated to the signal intensity, so an increase in magnetite content gives a stronger signal [98]. With these advantages, MNPs can be a potential label for clinical research, environmental and food safety testing. The portable Magnetic Assay Reader (MAR) (MagnaBioScience, Quantum Design, San Diego) was developed to interpret results. The tendency to associate in solutions because of the magnetic properties can be mentioned as a disadvantage. MNP conjugation with proteins is commonly performed by synthesizing iron-oxide (Fe3O4 or γ-Fe2O3) NPs, silica coating, surface modification with amino or carboxylic groups, and conjugation with biomolecules [99]. There are some examples of magnetic-based LFIA developed for clinical applications {e.g., tests for hCG hormone [100], cardiac troponin I [97], E. coli [101] human papillomavirus [102] and HIV antibodies [103]}.

5. Liposomes Liposomes are vesicles formed by a lipid bilayer with the hydrophobic chains of the lipids directed toward each other and the polar head-groups of the lipids oriented toward the extravesicular solution and the inner cavity. The size of the liposomes is in the ranges 50–800 nm. Usually, a reversed-phase evaporation method involving a water-in-oil emulsion is used to prepare the liposomes. A variety of phospholipids with different polar head-groups available for conjugation, reduction of liposome aggregation, and hydrophobic regions possessing different chain lengths and saturation are used to modify the properties of the resulting liposomes. Different chemicallyactive groups (including active groups for coupling biomolecules) can easily be incorporated on the liposome surface. It is important to mention that the properties and the amount of such groups can be varied over a wide range. This enables control of properties (e.g., the size of the liposomes and the surface density of covalently-bound biomolecules). The structure of liposomes offers the possibility of loading multiple signal-generating molecules. Molecules can associate with liposomes in several ways, including encapsulation within the aqueous inner cavity, partitioning within the lipid tails of the bilayer, and covalent and electrostatic interactions with the polar head-groups of the lipids [104]. A wide variety of molecules and NPs can be encapsulated within liposomes – enzymes, visual/colored and fluorescent dyes, QDs, and electrochemical and chemiluminescent markers. Because of the ability to encapsulate very high amounts of such labels, the detectable analyte concentration can be reduced by 2–3 orders of magnitude [105]. Comparison of biotin-enzyme-tagged liposomes with a biotin-tagged enzyme showed a 100–1000-fold increase in signal with liposomes for different immunosystems and different ways of signal generation [104]. A 500-fold lower LOD was found measuring the fluorescence of lysed dye-encapsulated liposomes versus a single-labeled probe [106]. Liposome advantages are long-term stability of the encapsulated signaling molecules and ease of labeling through direct incorporation of hydrophobically-modified nucleic-acid probes into their lipid bilayers [106]. However, in most cases, the enhancement was not as significant as would be expected because of steric hindrance and multivalency. Liposomes have many biorecognition elements on their surfaces, so one liposome can theoretically bind to several targets. Application of liposomes for analysis was reviewed by Edwards and Baeumner [104]. Lysing of the liposomes after completion of the immunochemical reaction is where liposomes can be differentiated from most of the other multi-loading carriers (e.g., in the case of lysing, the signal amplification is a result of liposome destruction with detergents). A drawback of liposomes is the difficulty of drying and subsequently recovering the liposomes without loss of their properties. This makes them hard to adapt to dry-chemistry assays (e.g., LFIA). However, some examples of lateral-flow tests were described. For 13   

application in lateral-flow tests, they could be targeted with DNA [106], RNA [107], and antigens [108–110]. Liposomes were used for multi-analyte detection by multi-spot format for cross-reacting analytes [107]. Lateral-flow tests were mainly developed toward high-molecular-weight analytes, but some examples were described for low-molecular-weight molecules {e.g., aflatoxin B1 [108]}. Variation of dyes could provide differently-colored liposome labels – methyl blue gives the liposomes a blue color [110], sulforhodamine B makes liposomes pink [106], and this latter could also be used for fluorescent measurement [107,109]. Aequorin is a photoprotein isolated from luminescent jellyfish in liposomes and it was also applied as a luminescent label [111]. For visual dyes, the color intensity may be measured semi-quantitatively by visual examination. However application of readers (e.g., QuadScan reflectance photometer, KGW Enterprises, Inc., Elkhart, IN, USA) can provide more accurate quantitative results.

5. Conclusions and outlook Research and applications in the field of rapid tests are at the crossroads of nanosciences, immunochemistry and biochemical techniques. Future development of rapid immunotests will profit from the continual advances in nanosciences, particularly in the production of new inorganic NPs with improved physical and chemical properties and stability. As NPs and biomolecules typically have the same nanometer scale, their conjugation will contribute to the establishment of novel techniques. Fast progress can be expected here, because advanced nanomaterials originally developed for materials research, physics and biology are already being used in immunosensing and bio-imaging and could provide readily-available approaches for rapid development of screening methods. While in most of the reported applications, NPs are seen as immunoassay labels achieving higher sensitivities and reducing the matrix influence, their application for multiplexing is only at the beginning. Moreover, the prospects of luminescent labels are not realized yet and more investigation is needed in this area. One area with prospects is the development of commerciallyavailable, hand-held, sensitive readers for evaluation of quantitative results and the integration into systems designed to optimize the performance of the overall assay from sample to answer. Acknowledgements This research was supported by The Russian Foundation of Basic Research (RFBR, Project 1203-91167) and by The Special Research Fund (BOF), Ghent University (01SB2510). References [1] Y.M. Kim, S.W. Oh, S.Y. Jeong, D.J. Pyo, E.Y. Choi, Environ. Sci. Technol. 37 (2003). 1899. [2] S. Girotti, S. Eremin, A. Montoya, M.J. Moreno, P. Caputo, M. D’Elia, L. Ripani, F.S. Romolo, E. Maiolini, Anal. Bioanal. Chem. 396 (2010) 687. [3] Y.R. Guo, S.Y. Liu, W.J. Gui, G.N. Zhu Anal. Biochem. 389 (2009) 32. [4] G.A. Posthuma-Trumpie, J. Korf, A. van Amerongen, Anal. Bioanal. Chem. 393 (2009) 569. [5] A. Warsinke, Anal. Bioanal. Chem. 393 (2009) 1393. [6] M. Seydack, Biosens. Bioelectron. 20 (2005) 2454. [7] C. Chafer-Pericas, A. Maquieira, R. Puchades, Trends Anal. Chem. 31 (2012) 144. [8] D. Pyo, J. Yoo, Immunoass. Immunochem. 33 (2012) 203. [9] Z. Wang, L.N. Ma, Coord. Chem. Rev. 253 (2009) 1607. [10] L.A. Dykman, V.A. Bogatyrev, Russ. Chem. Rev. 76 (2007) 199. [11] P. Bao, A.G. Frutos, C. Greef, J. Lahiri, U. Muller, T.C. Peterson, L. Warden, X. Xie, Anal. Chem. 74 (2002) 1792. 14   

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Table 1. Advantages and disadvantages of colored and luminescent nanoparticles applied as labels in immunoassay Label type Advantages Disadvantages Colored nanoparticles Colloidal • Biocompatibility • Insufficient brightness of standard gold • Possibility of modification to obtain spherical nanoparticles brighter labels with the same or different color • Possibility of developing procedures for application to obtain brighter color • Various readers are available • No functional groups on the Colloidal • Easy “gray pixel” processing because of surface carbon “black-on-white” test results • Physical adsorption of • Inexpensive production immunoreagents • Production easily scaled up • Polydisperse particles • Relatively low density of large particles • Possible presence of large allows stable suspension irregularly shaped particles in batches “Colloidal” • Higher brightness compared to molecular • dyes dyes Luminescent nanoparticles “Colloidal” • Higher fluorescence intensity compared • Cross-talk between excitation and fluorescent to molecular dyes emission signals dyes Infrared • Spectral elimination interferences from • No possibility for visual detection emitters biological fluorescent background • No commercially available readers • Sub-micron size is quite large relative to the typical protein size • Large size distribution • Long lifetime Up• Non-specific binding • Sequential two-photon absorption converting • Relatively low quantum efficiency possible in microsecond time scale emitters • Sub-micron size is quite large • Anti-Stokes luminescence minimizes relative to the typical protein size background (noise) • Insufficiently stable without • No photodegradation of biomolecules surface modification because of excitation in infrared area • Different combinations of rare-earth emitting and absorbing ions create more than 20 unique compositions • Easily produced from commerciallyavailable bulk material • Relatively low quantum efficiency Nanoparticl • Separates emission from background through lifetime differences • No simple and low-cost es with instruments for time-resolved long-living measurements emission Quantum • Small size • Toxicity dots • Intense fluorescence • Wide excitation and narrow emission bands   18   

Highlights Developments in application of nanoparticles as labels for rapid immunotests Colored and luminescent nanolabels are discussed in detail The possibility of simultaneous detection of multiple analytes and reader application  

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