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Cross-talk-free simultaneous fluoroimmunoassay of two biomarkers based on dual-color quantum dots
Analytica Chimica Acta 698 (2011) 44–50
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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Cross-talk-free simultaneous fluoroimmunoassay of two biomarkers based on dual-color quantum dots Zhijuan Cao a , Huan Li a , Choiwan Lau a,∗ , Yuhao Zhang b,∗∗ a b
School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, PR China Department of Neurology, Zhongshan Hospital, 180 Fenglin Road, Shanghai 200032, PR China
a r t i c l e
i n f o
Article history: Received 12 December 2010 Received in revised form 22 April 2011 Accepted 25 April 2011 Available online 5 May 2011 Keywords: Simultaneous detection Two biomarkers Polystyrene microspheres Quantum dots Cross-talk-free
a b s t r a c t In this article, we demonstrate the fabrication and simultaneous fluorescent detection of two biomarkers related to lung cancer. Polystyrene microspheres (PSM) were introduced as biomolecular immobilizing carriers and a 96-well filter plate was used as the separation platform. The whole experiment could be effectively carried out in a homogeneous system, as exemplified by the detection of carcinoembryonic antigen (CEA) and neuron specific enolase (NSE). First, two capture antibodies for CEA and NSE were immobilized on the PSM surface. Next, they reacted successively with two antigens and two modified detection antibodies. Finally, these two biomarkers could be recognized by streptavidin-conjugated quantum dots (QD) and goat-anti-FITC conjugated QD with a detection limit of 0.625 ng mL−1 , which was lower than the clinical cut-off level. The protocol showed good precision within 6.36% and good recovery in the range of 90.86–105.02%. Compared with several other assay formats reported previously, our new technique is competitive or even better. Furthermore, the immunosensor was successfully illustrated in 20 serum samples. Overall, this new immunoassay offers a promising alternative for the detection of biomarkers related to cancer diseases, taking advantage of simplicity, specificity, sensitivity and cost-efficiency. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Cancers are considered the main causes of death, and lung cancer is at the top of the list for both men and women, with a 5-year survival rate of less than 16% [1]. In recent years, protein biomarkers have shown great potential in the early diagnosis and treatment planning of cancer in its development through screening as well as staging, metastasis evaluation, and response to pharmacologic intervention [2]. Both monoplexed and multiplexed assays for biomarker detection have been developed [3–8]. Unlike the monoplexed assay, the multiplexed assay permits the simultaneous determination of multiple biomarkers within a single sample, requiring less sample, fewer repetitions of tedious procedures, reduced analysis time and lower cost per test. Commonly, two strategies have been applied for multiplexed assays. One is “planar arrays” (protein microarrays). The other one is “suspension arrays” (encoded microcarrier protocols), which offer several attractive aspects, such as improved solution kinetics, ease of assay modification and better quality control by batch
∗ Corresponding author. Tel.: +86 21 5198 0028; fax: +86 21 5198 0028. ∗∗ Corresponding author. Tel.: +86 21 6404 1900x2921; fax: +86 21 5198 0028. E-mail addresses: [email protected] (C. Lau), [email protected] (Y. Zhang). 0003-2670/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2011.04.045
synthesis [9]. Methods suitable for the multiplexed assays include electrochemistry [10], chemiluminescence (CL) [11–16], Raman spectroscopy [17], SPR [18], scannometry [19], and fluorescence (FL) [9,20–23], etc. among which spectral multiplexing (multicolor signaling) is the most common protocol. However, spectral multiplexing with organic dyes is mostly limited by their comparatively broad emission bands of slightly structured shape and the narrow wavelength region of optimal excitation of each dye. Quantum dots (QD) are emerging as new kinds of fluorescent tag. Compared with organic fluorophores, QD provide tunable, symmetrical and narrow emission bands with high photostability. More significantly, different-sized QD can be excited with a single wavelength excitation light [24–26]. Therefore, QD are considered one of the most promising materials in multiplexed assays [27–32]. Herein, a new cross-talk-free duplex fluoroimmunoassay for cancer-related biomarkers was developed using multiple QD as detection elements. A fast homogeneous immunoreaction as well as a simple heterogeneous separation process was achieved by the coupling of the submicrometer-sized polystyrene microspheres (PSM) as the carrier and the 96-well filter plate as the reaction and separation container. After the antibody-antigen interaction, the PSM conjugates can be separated simply and rapidly by a vacuum filter. To demonstrate our original idea, we applied this novel fluoroimmunoassay for the measurement of two lung cancer biomarkers, carcinoembryonic antigen (CEA) and neuron specific enolase (NSE) [33].
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2. Materials and methods
2.5. Single and duplex fluoroimmunoassay procedure
2.1. Chemicals and materials
In a typical experiment, PSM were firstly activated by EDC in IM buffer for 20 min. And then the CEA or NSE capture antibody was immobilized onto the activated PSM surface at 37 ◦ C for 40 min, which was washed with IM buffer for three times. Excess antibody was removed by decanting the supernatant, and the precipitation cake was resuspended and blocked with blocking buffer (PBS containing 5% BSA, 50 L per sample) at 37 ◦ C for 30 min. After decantation of blocking buffer, CEA and NSE capture antibody-PSM conjugates were mixed (or not for single-biomarker assay), and then divided into the appropriate wells in the 96-well filter plate with 200 L PBSTW. The PSM conjugates were washed for three times using the vacuum filter, taking only 20 s per wash. Serial dilutions of antigens (single or a mixture) in PBSTW-2% BSA (w/v, 100 L per well) were made and applied to the above wells respectively, and allowed to shake gently at 37 ◦ C for 60 min. After that, the wells were washed 3 times with PBSTW by vacuum filtration. Then 50 L of modified CEA detection antibody and NSE detection antibody with the optimal concentration (alone or in mixture) were added into the above wells and incubated in PBS-2% BSA at 37 ◦ C for 30 min, followed by the binding of SA-QD and anti-FITCQD (alone or in mixture) in PBS with 2% BSA at 37 ◦ C for another 30 min. Unbound QD conjugates were drawn out of the wells by vacuum filtration and the resultant PSM conjugates were washed thoroughly with PBSTW, followed by the transfer into a 96-well black microplate with 150 L of PBS and FL measurement at emissions of 585 nm and 655 nm, respectively. All experiments were performed at least triplicate.
Monoclonal capture and detection antibodies for CEA and NSE were purchased from Shanghai Linc Bio Science Co. China. CEA and NSE antigens, bovine serum albumin (BSA), fluorescein isothiocyanate (FITC) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were obtained from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from GIBCO (Grand island, NY, USA). Streptavidin-conjugated QD (SA-QD, 585 nm) and goat-anti-FITC conjugated QD (anti-FITC-QD, 655 nm) and succinimidyl-6-(biotinamido) hexanoate (NHS-LC-biotin) were purchased from Invitrogen (Carlsbad, CA, USA). Carboxylated PSM (26 mg mL−1 , diameter of 500 nm, 1% solids) was obtained from Polyscience Inc. (Warrington PA, USA). All the other reagents were commercially available analytical grade. Normal individual serum (NIS) and patient serum samples were offered by Zhongshan Hospital, Shanghai, China.
2.2. Buffers All solutions were prepared with deionized water from a Millipore system (Millipore XQ). Buffer SCB was 25 mM sodium carbonate buffer, pH 9.2; IM buffer was 0.1 M imidazole, pH 6.0; PBS consisted of 8.60 g NaCl, 2.38 g K2 HPO4, and 0.286 g NaH2 PO4 in 1 L pure water, pH 7.4; PBSTW was PBS containing 0.05% Tween 20.
2.6. Sample analysis 2.3. Apparatus FL experiments were all performed using an F-7000 fluorescence spectrophotometer (HITACHI. Ltd., Japan) controlled by FL Solution software for curve-fitting and peak height determination. All excitation and emission slits were set at 5 nm and 10 nm, respectively. The excitation was settled at 355 nm with emissions of 585 nm and 655 nm. Absorbance was determined by a U-2900 Spectrophotometer (HITACHI. Ltd., Japan). All reactions were conducted in air-bath shaker (MaxQ 4000, Thermo Scientific, Iowa, USA). An AcroWell 96 filter plate with 0.2 m of GHP membrane at the well bottom and vacuum manifold were purchased from Pall Corporation.
For serum matrix effect, serial concentrations of antigens were diluted in PBSTW-2% BSA media containing either normal individual serum or FBS. For serum analysis, calibration curves were constructed by diluting standard antigens in the PBSTW-2% BSA-50% FBS solution. To evaluate recoveries, 50 L of each normal individual sample was mixed with 50 L of CEA and NSE standard solution in PBSTW-4% BSA at designed concentrations (10, 40, 160 ng mL−1 ). Each patient serum sample was diluted in PBSTW-4% BSA by 1:1 (v/v). And all of the prepared calibrators and sample solutions were conducted as the same procedures described in Section 2.5. 3. Results and discussion
2.4. Preparation of modified detection antibodies for duplex fluoroimmunoassays Typically, CEA detection antibody with the concentration of 1 mg mL−1 was dissolved in PBS. An appropriate volume of NHS-LCbiotin (10 mM in DMF), 20-fold molar excess of protein, was added and then incubated at room temperature for 30 min. The resultant CEA detection solution (biotinylated CEA detection antibody) was dialyzed overnight in 500 mL PBS at 4 ◦ C to remove non-reacted biotin, and then stored at −20 ◦ C before use (0.96 mg mL−1 detected by UV). 1 mL of NSE detection antibody solution (2 mg mL−1 ) was first concentrated to 10 mg mL−1 using 50 kDa molecular weight cutoff centrifuge concentrators (Millipore, USA). Then, 0.8 mL of FITC solution (1 g in buffer SCB) was added and the solution was incubated at room temperature for 30 min. The resultant NSE detection antibody solution (FITC-tagged NSE detection antibody) was first dialyzed in 25 mM SCB (1 L) and then overnight in PBS (1 L). Finally, the modified NSE detection antibody solution was stored at −20 ◦ C before use (1.96 mg mL−1 detected by UV).
A “sandwich-type” detection strategy was employed in our design (Scheme 1) and two biomarkers associated with lung cancer were determined simultaneously. Prior to the binding of capture antibodies, the carboxylated PSM was activated by EDC. As a result, the capture antibodies brought the special antigens, along with reporter antibodies and QD conjugates proximal to the PSM surface. The sandwich complex thus formed only in the presence of the appropriate antigen. In addition, each color QD conjugate was capped by one specific linker molecule, streptavidin or goatanti-FITC antibody, leading to their special identification of the corresponding detection antibodies, i.e. biotinylated CEA detection antibody and FITC-tagged NSE detection antibody. After that, FL signal was recorded by the FL spectrophotometer. As seen in Fig. 1, there was minimal cross-talk at each emission peak of 585 nm and 655 nm with 1:2000 dilutions of QD in PBS, demonstrating that two biomarkers could be measured simultaneously. Additionally, several parameters were optimized systematically for this novel duplex fluoroimmunoassay, including the amounts of EDC, carboxylated PSM, two capture antibodies, modified detection antibodies and QD conjugates, etc.
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Scheme 1. Schematic representation of dual-color QD-based fluoroimmunoassay.
3.1. Optimization of EDC concentration
3.2. Optimization of CEA sensing conditions
Carboxylated PSM should first be activated by EDC, and then bound with capture antibodies. Thus, several EDC concentrations were investigated (in Fig. 2). FL intensity maintained an approximately steady level for EDC concentrations between 0.6 and 4.8 mg mL−1 , and then decreased as the EDC concentration was increased in the range of 4.8–9.6 mg mL−1 . It was suggested that this decrease was associated with PSM aggregation due to excessive activation, as was visually observed. Thus, a 2.4 mg mL−1 concentration of EDC was used in subsequent work.
Various parameters for CEA assay were explored. As shown in Fig. 3A, 26, 52, 78, 104 and 130 g of PSM were studied. FL intensity was observed to initially increase with increasing PSM amount, reaching a maximum at 78 g, and then decreasing. It was postulated that an excess of activated PSM led to the decrease of capture antibody on a single microsphere, which was not beneficial to bind CEA antigen. Thus, 78 g of PSM was used in subsequent experiments for CEA detection. The effect of the amount of CEA capture antibody was subsequently examined and optimized. As described in Fig. 3B, FL intensity was increased in the range of 20–100 g mL−1 of CEA capture antibodies and then decreased slowly. This decrease was attributed to steric and electrostatic hindrances, arising from more tightly packed capture antibody on the surface of activated PSM, limiting access to the surface-bound capture antibody by CEA antigen. Hence, subsequent study employed 100 g mL−1 capture antibody. After that, CEA detection antibody (Fig. 3C) and
Fig. 1. Emission spectra of the two QD conjugates with the dilution of 1:2000 used in the multianalyte assays. Data were collected using the HITACHI F-7000 fluorescence spectrophotometer. An excitation of 355 nm was used. () QD with the emission peak at 655 nm, ( ) QD mixture with the emission peak at both 585 and 655 nm, ( ) QD with the emission peak at 585 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Fig. 2. FL intensity vs concentration of EDC. Experimental conditions: 78 g of PSM, 100 g mL−1 of NSE capture antibody, 20 ng mL−1 of NSE, 5 g mL−1 of NSE detection antibody, 1:1000 diluted anti-FITC-QD.
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Fig. 3. FL intensity vs amount of PSM (A), CEA capture antibody (B), biotinylated CEA detection antibody (C) and dilution of SA-QD (D). Experimental conditions: (A) 60 g mL−1 of CEA capture antibody, 20 g mL−1 of CEA detection antibody, 1:600 of SA-QD; (B) 78 g of PSM, 20 g mL−1 of CEA detection antibody, 1:600 of SA-QD; (C) 78 g of PSM, 100 g mL−1 of CEA capture antibody, 1:600 of SA-QD; (D) 78 g of PSM, 100 g mL−1 of CEA capture antibody and 40 g mL−1 of CEA detection antibody. All experiments were done at least triplicate with 20 ng mL−1 of CEA antigen.
SA-QD dilution (Fig. 3D) were examined successively. The optimal FL intensity was obtained at 40 g mL−1 of CEA detection antibody and 1:600 of SA-QD, which were then selected for the following experiments.
intensity by using from 52 to 104 g of PSM), 60 g mL−1 of NSE capture antibody, 10 g mL−1 of NSE detection antibody, and 1:400 of anti-FITC-QD (Fig. 4A–D).
3.4. Performance of the duplexed fluoroimmunoassay 3.3. Optimization of NSE sensing conditions Similarly, NSE responsive sensors were prepared with NSE capture and FITC-tagged detection antibodies and anti-FITC-QD. A series of parameters were investigated to establish optimal conditions for NSE detection. As a result, optimized parameters were found to be 78 g of PSM (there was no obvious difference for FL
Under the above-optimized conditions, Fig. 5 illustrated that mixtures of two antigens could be analyzed in a quantitative fashion. Firstly according to the FL spectra (Fig. 5A), the FL intensity was positively correlative with CEA and NSE concentration. Nevertheless, it was noteworthy that the baseline had a slight decrease in the range of 550–620 nm, which was caused by the PSM carrier and
Fig. 4. FL intensity vs amount of PSM (A), NSE capture antibody (B), FITC-tagged NSE detection antibody (C) and the dilution of anti-FITC-QD (D). Experimental conditions: (A) 60 g mL−1 of NSE capture antibody, 20 g mL−1 of NSE detection antibody, 1:600 of anti-FITC-QD; (B) 78 g of PSM, 20 g mL−1 of NSE detection antibody, 1:600 of anti-FITC-QD; (C) 78 g of PSM, 60 g mL−1 of NSE capture antibody, 1:600 of anti-FITC-QD; (D) 78 g of PSM, 60 g mL−1 of NSE capture antibody and 10 g mL−1 of NSE detection antibody. Each parameter was done at least triplicate with 20 ng mL−1 of NSE antigen.
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Fig. 5. Performance of dual-biomarker fluoroimmunoassay. (A) Scan of wells with two analytes immobilized on a surface containing capture antibodies for both analytes and detected using a mixture of SA-QD and anti-FITC-QD conjugates. Measured values were shown as different color circles. (B) Calibration curve of the assay for CEA with/without NSE at the emission wavelength of 585 nm. (C) Calibration curve of the assay for NSE with/without CEA at the emission wavelength of 655 nm. Each data point represents the average of at least three independent measurements.
did not affect the analysis. In addition, this duplex assay showed no obvious difference with single-analyte assays, as also demonstrated in Fig. 5B and C. FL intensities for mixtures of CEA and NSE as well as individual antigen were practically the same, and confirmed that there was no obvious cross reaction among these two antibody-antigen interactions. Furthermore, some lung cancer drugs such as adriamycin and carboplatin were also studied. The results showed that these drugs did not provide any interference. It was supposed that the drugs could not be identified and bound by the specific antibodies of two biomarkers. FL signals were in response to sample mixtures containing increasing levels of two lung cancer-related biomarkers. A calibration graph in the concentration range of 1.25–80 ng mL−1 showed a good linear correlation between the FL intensity and CEA in the presence and absence of NSE, represented by I = 1.26C − 3.78 (R2 = 0.9926)
for individual CEA and I = 1.24C − 2.89 (R2 = 0.9951) for a mixture (as illustrated in Fig. 5B). Fig. 5 C showed the relationship between the FL intensity and NSE in the presence and absence of CEA. A calibration graph of NSE in the range of 1.25–80 ng mL−1 was represented by I = 1.21N + 0.06 (R2 = 0.9992) for individual CEA and I = 1.20N + 0.59 (R2 = 0.9997) for a mixture. Both detection limits for two biomarkers were estimated to be 0.625 ng mL−1 . The precisions (2.5, 10 and 40 ng mL−1 ) were found to be 2.23–5.13% for CEA and 0.09–6.36% for NSE. In the presence of 40 ng mL−1 NSE, the recoveries of CEA (2.5, 10 and 40 ng mL−1 ) were 104.05, 100.03 and 99.29%, respectively. In the presence of 40 ng mL−1 CEA, the recoveries for NSE were calculated to be 90.86, 99.11 and 105.02%. Overall, our new technique allowed detection of two lung cancer markers down to subnanogram per milliliter level and was competitive with or even better than other assay formats (Table 1).
Table 1 Comparison of different multiplexed immunoassays. Analytical methods
Number of analytes
Label
Detection of limit
FL microarray Electrochemical immunoassay (ECL) Fluorescent immunoassay (FL) CL immunoassay CL immunoassay SERS Capacitive immunosensor CL immunoassay FL immunoassay CL immunoassay FL immunoassay FL immunoassay FL immunoassay ECL immunoassay Colorimetric immunoassay ICP-MS immunoassay ICP-MS immunoassay ECL immunoassay FL immunoassaya
6 4 4 4 4 3 3 3 3 2 2 2 2 2 2 2 2 2 2
FITC Colloidal nanocrystals QDs ALP ALP and HRP Silver nanoparticles Label-free ALP Alexa Fluor HRP QDs QDs QDs QDs HRP Eu3+ and Sm3+ Eu3+ and gold nanoparticles Gold nanoparticle QDs
20 ng mL−1 or 40 ng mL−1 [22] 10 ng mL−1 [10] – [27] 0.52 g L−1 , 0.49 kU L−1 , 0.79 kU L−1 , and 0.55 g L−1 [12] 0.15, 0.80, and 2.0 U mL−1 and 0.65 ng mL−1 [15] 100 pM [17] 25 pg mL−1 [34] 0.60 g L−1 , 0.080 g L−1 , and 0.70 kU L−1 [13] 0.2, 1 and 2 g mL−1 [35] 0.41 and 0.39 ng mL−1 [11] 1.3 and 1.2 ng mL−1 [31] 2.5 pM [32] 1 pM [29] 30 and 10 pg mL−1 [36] 0.02 ng mL−1 [37] 1.2 and 1.7 ng mL−1 [38] 2ng mL−1 [39] 1.0 and 1.5 ng mL−1 [40] 0.625 ng mL−1 (500 pM)
a
This work.
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Fig. 6. FL intensity of CEA and NSE in PBSTW-2% BSA medium containing different concentrations of NIS and FBS. Experimental conditions: CEA detection, 78 g PSM, 100 g mL−1 of NSE capture antibody, 40 g mL−1 of CEA detection antibody, and 1:600 of SA-QD; NSE detection, 78 g PSM, 60 g mL−1 of NSE capture antibody, 10 g mL−1 of NSE detection antibody, and 1:400 of anti-FITC-QD. The detection procedure was carried out as described in the Section 2.5.
Fig. 7. Comparison of CEA (A) and NSE (B) determinations in patient serum samples with the proposed method and commercial ELISA kits.
3.5. Application of the fluoroimmunoassay in human serum samples As shown in Fig. 6, FL intensity decreased with the increase of NIS, which was attributed to interference from NIS. Thus, different concentrations of FBS were investigated in parallel, and FL intensity was almost the same in the PBSTW-2% BSA medium containing equal percent of NIS or FBS (data of 30% FBS and NIS not shown). Therefore, PBSTW-2% BSA-50% FBS was employed to dilute and prepare the standard solutions for the construction of calibration curves and the analysis of real samples. The recoveries for 5, 20 and 80 ng mL−1 from 20 normal serum samples were calculated to be 95.01 ± 15.83%, 90.25 ± 18.61% and 94.63 ± 12.09% for CEA, respectively and 96.78 ± 16.53%, 102.30 ± 14.08% and 92.56 ± 6.40% for NSE, respectively. The assay results of clinical serum samples using the proposed method were compared with referred values obtained by commercial ELISA kits (data from Zhongshan Hospital). The results were shown in Fig. 7. Compared with the referred values, the good correlation was obtained (R2 > 0.90), indicating acceptable accuracy. 4. Conclusions A new duplex fluoroimmunoassay platform for the homogeneous determination of two biomarkers has been demonstrated. The optimal assay condition provides a good sensitivity to meet the requirement of cut-off levels and good reaction media for detection of clinical serum samples. Moreover, this new approach offers the following advantages: (1) two biomarkers can be detected in a single experiment with a single sample, consuming less time and sample. (2) Unlike the conventional biomarker detection assay, such as ELISA and heterogeneous fluoroimmunoassay, our new method employs PSM as the capture antibodies conjugated carrier to make the entire reaction occur in an almost homogeneous situation. (3) The wash procedure has been simplified by using a 96-well filter plate coupling with the vacuum filter device. Each wash can be finished within 20 s. (4) The FL signal derives from two cross-talk-free QD conjugated probes and easily obtained by
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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Cross-talk-free simultaneous fluoroimmunoassay of two biomarkers based on dual-color quantum dots Zhijuan Cao a , Huan Li a , Choiwan Lau a,∗ , Yuhao Zhang b,∗∗ a b
School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, PR China Department of Neurology, Zhongshan Hospital, 180 Fenglin Road, Shanghai 200032, PR China
a r t i c l e
i n f o
Article history: Received 12 December 2010 Received in revised form 22 April 2011 Accepted 25 April 2011 Available online 5 May 2011 Keywords: Simultaneous detection Two biomarkers Polystyrene microspheres Quantum dots Cross-talk-free
a b s t r a c t In this article, we demonstrate the fabrication and simultaneous fluorescent detection of two biomarkers related to lung cancer. Polystyrene microspheres (PSM) were introduced as biomolecular immobilizing carriers and a 96-well filter plate was used as the separation platform. The whole experiment could be effectively carried out in a homogeneous system, as exemplified by the detection of carcinoembryonic antigen (CEA) and neuron specific enolase (NSE). First, two capture antibodies for CEA and NSE were immobilized on the PSM surface. Next, they reacted successively with two antigens and two modified detection antibodies. Finally, these two biomarkers could be recognized by streptavidin-conjugated quantum dots (QD) and goat-anti-FITC conjugated QD with a detection limit of 0.625 ng mL−1 , which was lower than the clinical cut-off level. The protocol showed good precision within 6.36% and good recovery in the range of 90.86–105.02%. Compared with several other assay formats reported previously, our new technique is competitive or even better. Furthermore, the immunosensor was successfully illustrated in 20 serum samples. Overall, this new immunoassay offers a promising alternative for the detection of biomarkers related to cancer diseases, taking advantage of simplicity, specificity, sensitivity and cost-efficiency. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Cancers are considered the main causes of death, and lung cancer is at the top of the list for both men and women, with a 5-year survival rate of less than 16% [1]. In recent years, protein biomarkers have shown great potential in the early diagnosis and treatment planning of cancer in its development through screening as well as staging, metastasis evaluation, and response to pharmacologic intervention [2]. Both monoplexed and multiplexed assays for biomarker detection have been developed [3–8]. Unlike the monoplexed assay, the multiplexed assay permits the simultaneous determination of multiple biomarkers within a single sample, requiring less sample, fewer repetitions of tedious procedures, reduced analysis time and lower cost per test. Commonly, two strategies have been applied for multiplexed assays. One is “planar arrays” (protein microarrays). The other one is “suspension arrays” (encoded microcarrier protocols), which offer several attractive aspects, such as improved solution kinetics, ease of assay modification and better quality control by batch
∗ Corresponding author. Tel.: +86 21 5198 0028; fax: +86 21 5198 0028. ∗∗ Corresponding author. Tel.: +86 21 6404 1900x2921; fax: +86 21 5198 0028. E-mail addresses: [email protected] (C. Lau), [email protected] (Y. Zhang). 0003-2670/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2011.04.045
synthesis [9]. Methods suitable for the multiplexed assays include electrochemistry [10], chemiluminescence (CL) [11–16], Raman spectroscopy [17], SPR [18], scannometry [19], and fluorescence (FL) [9,20–23], etc. among which spectral multiplexing (multicolor signaling) is the most common protocol. However, spectral multiplexing with organic dyes is mostly limited by their comparatively broad emission bands of slightly structured shape and the narrow wavelength region of optimal excitation of each dye. Quantum dots (QD) are emerging as new kinds of fluorescent tag. Compared with organic fluorophores, QD provide tunable, symmetrical and narrow emission bands with high photostability. More significantly, different-sized QD can be excited with a single wavelength excitation light [24–26]. Therefore, QD are considered one of the most promising materials in multiplexed assays [27–32]. Herein, a new cross-talk-free duplex fluoroimmunoassay for cancer-related biomarkers was developed using multiple QD as detection elements. A fast homogeneous immunoreaction as well as a simple heterogeneous separation process was achieved by the coupling of the submicrometer-sized polystyrene microspheres (PSM) as the carrier and the 96-well filter plate as the reaction and separation container. After the antibody-antigen interaction, the PSM conjugates can be separated simply and rapidly by a vacuum filter. To demonstrate our original idea, we applied this novel fluoroimmunoassay for the measurement of two lung cancer biomarkers, carcinoembryonic antigen (CEA) and neuron specific enolase (NSE) [33].
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2. Materials and methods
2.5. Single and duplex fluoroimmunoassay procedure
2.1. Chemicals and materials
In a typical experiment, PSM were firstly activated by EDC in IM buffer for 20 min. And then the CEA or NSE capture antibody was immobilized onto the activated PSM surface at 37 ◦ C for 40 min, which was washed with IM buffer for three times. Excess antibody was removed by decanting the supernatant, and the precipitation cake was resuspended and blocked with blocking buffer (PBS containing 5% BSA, 50 L per sample) at 37 ◦ C for 30 min. After decantation of blocking buffer, CEA and NSE capture antibody-PSM conjugates were mixed (or not for single-biomarker assay), and then divided into the appropriate wells in the 96-well filter plate with 200 L PBSTW. The PSM conjugates were washed for three times using the vacuum filter, taking only 20 s per wash. Serial dilutions of antigens (single or a mixture) in PBSTW-2% BSA (w/v, 100 L per well) were made and applied to the above wells respectively, and allowed to shake gently at 37 ◦ C for 60 min. After that, the wells were washed 3 times with PBSTW by vacuum filtration. Then 50 L of modified CEA detection antibody and NSE detection antibody with the optimal concentration (alone or in mixture) were added into the above wells and incubated in PBS-2% BSA at 37 ◦ C for 30 min, followed by the binding of SA-QD and anti-FITCQD (alone or in mixture) in PBS with 2% BSA at 37 ◦ C for another 30 min. Unbound QD conjugates were drawn out of the wells by vacuum filtration and the resultant PSM conjugates were washed thoroughly with PBSTW, followed by the transfer into a 96-well black microplate with 150 L of PBS and FL measurement at emissions of 585 nm and 655 nm, respectively. All experiments were performed at least triplicate.
Monoclonal capture and detection antibodies for CEA and NSE were purchased from Shanghai Linc Bio Science Co. China. CEA and NSE antigens, bovine serum albumin (BSA), fluorescein isothiocyanate (FITC) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were obtained from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from GIBCO (Grand island, NY, USA). Streptavidin-conjugated QD (SA-QD, 585 nm) and goat-anti-FITC conjugated QD (anti-FITC-QD, 655 nm) and succinimidyl-6-(biotinamido) hexanoate (NHS-LC-biotin) were purchased from Invitrogen (Carlsbad, CA, USA). Carboxylated PSM (26 mg mL−1 , diameter of 500 nm, 1% solids) was obtained from Polyscience Inc. (Warrington PA, USA). All the other reagents were commercially available analytical grade. Normal individual serum (NIS) and patient serum samples were offered by Zhongshan Hospital, Shanghai, China.
2.2. Buffers All solutions were prepared with deionized water from a Millipore system (Millipore XQ). Buffer SCB was 25 mM sodium carbonate buffer, pH 9.2; IM buffer was 0.1 M imidazole, pH 6.0; PBS consisted of 8.60 g NaCl, 2.38 g K2 HPO4, and 0.286 g NaH2 PO4 in 1 L pure water, pH 7.4; PBSTW was PBS containing 0.05% Tween 20.
2.6. Sample analysis 2.3. Apparatus FL experiments were all performed using an F-7000 fluorescence spectrophotometer (HITACHI. Ltd., Japan) controlled by FL Solution software for curve-fitting and peak height determination. All excitation and emission slits were set at 5 nm and 10 nm, respectively. The excitation was settled at 355 nm with emissions of 585 nm and 655 nm. Absorbance was determined by a U-2900 Spectrophotometer (HITACHI. Ltd., Japan). All reactions were conducted in air-bath shaker (MaxQ 4000, Thermo Scientific, Iowa, USA). An AcroWell 96 filter plate with 0.2 m of GHP membrane at the well bottom and vacuum manifold were purchased from Pall Corporation.
For serum matrix effect, serial concentrations of antigens were diluted in PBSTW-2% BSA media containing either normal individual serum or FBS. For serum analysis, calibration curves were constructed by diluting standard antigens in the PBSTW-2% BSA-50% FBS solution. To evaluate recoveries, 50 L of each normal individual sample was mixed with 50 L of CEA and NSE standard solution in PBSTW-4% BSA at designed concentrations (10, 40, 160 ng mL−1 ). Each patient serum sample was diluted in PBSTW-4% BSA by 1:1 (v/v). And all of the prepared calibrators and sample solutions were conducted as the same procedures described in Section 2.5. 3. Results and discussion
2.4. Preparation of modified detection antibodies for duplex fluoroimmunoassays Typically, CEA detection antibody with the concentration of 1 mg mL−1 was dissolved in PBS. An appropriate volume of NHS-LCbiotin (10 mM in DMF), 20-fold molar excess of protein, was added and then incubated at room temperature for 30 min. The resultant CEA detection solution (biotinylated CEA detection antibody) was dialyzed overnight in 500 mL PBS at 4 ◦ C to remove non-reacted biotin, and then stored at −20 ◦ C before use (0.96 mg mL−1 detected by UV). 1 mL of NSE detection antibody solution (2 mg mL−1 ) was first concentrated to 10 mg mL−1 using 50 kDa molecular weight cutoff centrifuge concentrators (Millipore, USA). Then, 0.8 mL of FITC solution (1 g in buffer SCB) was added and the solution was incubated at room temperature for 30 min. The resultant NSE detection antibody solution (FITC-tagged NSE detection antibody) was first dialyzed in 25 mM SCB (1 L) and then overnight in PBS (1 L). Finally, the modified NSE detection antibody solution was stored at −20 ◦ C before use (1.96 mg mL−1 detected by UV).
A “sandwich-type” detection strategy was employed in our design (Scheme 1) and two biomarkers associated with lung cancer were determined simultaneously. Prior to the binding of capture antibodies, the carboxylated PSM was activated by EDC. As a result, the capture antibodies brought the special antigens, along with reporter antibodies and QD conjugates proximal to the PSM surface. The sandwich complex thus formed only in the presence of the appropriate antigen. In addition, each color QD conjugate was capped by one specific linker molecule, streptavidin or goatanti-FITC antibody, leading to their special identification of the corresponding detection antibodies, i.e. biotinylated CEA detection antibody and FITC-tagged NSE detection antibody. After that, FL signal was recorded by the FL spectrophotometer. As seen in Fig. 1, there was minimal cross-talk at each emission peak of 585 nm and 655 nm with 1:2000 dilutions of QD in PBS, demonstrating that two biomarkers could be measured simultaneously. Additionally, several parameters were optimized systematically for this novel duplex fluoroimmunoassay, including the amounts of EDC, carboxylated PSM, two capture antibodies, modified detection antibodies and QD conjugates, etc.
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Scheme 1. Schematic representation of dual-color QD-based fluoroimmunoassay.
3.1. Optimization of EDC concentration
3.2. Optimization of CEA sensing conditions
Carboxylated PSM should first be activated by EDC, and then bound with capture antibodies. Thus, several EDC concentrations were investigated (in Fig. 2). FL intensity maintained an approximately steady level for EDC concentrations between 0.6 and 4.8 mg mL−1 , and then decreased as the EDC concentration was increased in the range of 4.8–9.6 mg mL−1 . It was suggested that this decrease was associated with PSM aggregation due to excessive activation, as was visually observed. Thus, a 2.4 mg mL−1 concentration of EDC was used in subsequent work.
Various parameters for CEA assay were explored. As shown in Fig. 3A, 26, 52, 78, 104 and 130 g of PSM were studied. FL intensity was observed to initially increase with increasing PSM amount, reaching a maximum at 78 g, and then decreasing. It was postulated that an excess of activated PSM led to the decrease of capture antibody on a single microsphere, which was not beneficial to bind CEA antigen. Thus, 78 g of PSM was used in subsequent experiments for CEA detection. The effect of the amount of CEA capture antibody was subsequently examined and optimized. As described in Fig. 3B, FL intensity was increased in the range of 20–100 g mL−1 of CEA capture antibodies and then decreased slowly. This decrease was attributed to steric and electrostatic hindrances, arising from more tightly packed capture antibody on the surface of activated PSM, limiting access to the surface-bound capture antibody by CEA antigen. Hence, subsequent study employed 100 g mL−1 capture antibody. After that, CEA detection antibody (Fig. 3C) and
Fig. 1. Emission spectra of the two QD conjugates with the dilution of 1:2000 used in the multianalyte assays. Data were collected using the HITACHI F-7000 fluorescence spectrophotometer. An excitation of 355 nm was used. () QD with the emission peak at 655 nm, ( ) QD mixture with the emission peak at both 585 and 655 nm, ( ) QD with the emission peak at 585 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Fig. 2. FL intensity vs concentration of EDC. Experimental conditions: 78 g of PSM, 100 g mL−1 of NSE capture antibody, 20 ng mL−1 of NSE, 5 g mL−1 of NSE detection antibody, 1:1000 diluted anti-FITC-QD.
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Fig. 3. FL intensity vs amount of PSM (A), CEA capture antibody (B), biotinylated CEA detection antibody (C) and dilution of SA-QD (D). Experimental conditions: (A) 60 g mL−1 of CEA capture antibody, 20 g mL−1 of CEA detection antibody, 1:600 of SA-QD; (B) 78 g of PSM, 20 g mL−1 of CEA detection antibody, 1:600 of SA-QD; (C) 78 g of PSM, 100 g mL−1 of CEA capture antibody, 1:600 of SA-QD; (D) 78 g of PSM, 100 g mL−1 of CEA capture antibody and 40 g mL−1 of CEA detection antibody. All experiments were done at least triplicate with 20 ng mL−1 of CEA antigen.
SA-QD dilution (Fig. 3D) were examined successively. The optimal FL intensity was obtained at 40 g mL−1 of CEA detection antibody and 1:600 of SA-QD, which were then selected for the following experiments.
intensity by using from 52 to 104 g of PSM), 60 g mL−1 of NSE capture antibody, 10 g mL−1 of NSE detection antibody, and 1:400 of anti-FITC-QD (Fig. 4A–D).
3.4. Performance of the duplexed fluoroimmunoassay 3.3. Optimization of NSE sensing conditions Similarly, NSE responsive sensors were prepared with NSE capture and FITC-tagged detection antibodies and anti-FITC-QD. A series of parameters were investigated to establish optimal conditions for NSE detection. As a result, optimized parameters were found to be 78 g of PSM (there was no obvious difference for FL
Under the above-optimized conditions, Fig. 5 illustrated that mixtures of two antigens could be analyzed in a quantitative fashion. Firstly according to the FL spectra (Fig. 5A), the FL intensity was positively correlative with CEA and NSE concentration. Nevertheless, it was noteworthy that the baseline had a slight decrease in the range of 550–620 nm, which was caused by the PSM carrier and
Fig. 4. FL intensity vs amount of PSM (A), NSE capture antibody (B), FITC-tagged NSE detection antibody (C) and the dilution of anti-FITC-QD (D). Experimental conditions: (A) 60 g mL−1 of NSE capture antibody, 20 g mL−1 of NSE detection antibody, 1:600 of anti-FITC-QD; (B) 78 g of PSM, 20 g mL−1 of NSE detection antibody, 1:600 of anti-FITC-QD; (C) 78 g of PSM, 60 g mL−1 of NSE capture antibody, 1:600 of anti-FITC-QD; (D) 78 g of PSM, 60 g mL−1 of NSE capture antibody and 10 g mL−1 of NSE detection antibody. Each parameter was done at least triplicate with 20 ng mL−1 of NSE antigen.
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Fig. 5. Performance of dual-biomarker fluoroimmunoassay. (A) Scan of wells with two analytes immobilized on a surface containing capture antibodies for both analytes and detected using a mixture of SA-QD and anti-FITC-QD conjugates. Measured values were shown as different color circles. (B) Calibration curve of the assay for CEA with/without NSE at the emission wavelength of 585 nm. (C) Calibration curve of the assay for NSE with/without CEA at the emission wavelength of 655 nm. Each data point represents the average of at least three independent measurements.
did not affect the analysis. In addition, this duplex assay showed no obvious difference with single-analyte assays, as also demonstrated in Fig. 5B and C. FL intensities for mixtures of CEA and NSE as well as individual antigen were practically the same, and confirmed that there was no obvious cross reaction among these two antibody-antigen interactions. Furthermore, some lung cancer drugs such as adriamycin and carboplatin were also studied. The results showed that these drugs did not provide any interference. It was supposed that the drugs could not be identified and bound by the specific antibodies of two biomarkers. FL signals were in response to sample mixtures containing increasing levels of two lung cancer-related biomarkers. A calibration graph in the concentration range of 1.25–80 ng mL−1 showed a good linear correlation between the FL intensity and CEA in the presence and absence of NSE, represented by I = 1.26C − 3.78 (R2 = 0.9926)
for individual CEA and I = 1.24C − 2.89 (R2 = 0.9951) for a mixture (as illustrated in Fig. 5B). Fig. 5 C showed the relationship between the FL intensity and NSE in the presence and absence of CEA. A calibration graph of NSE in the range of 1.25–80 ng mL−1 was represented by I = 1.21N + 0.06 (R2 = 0.9992) for individual CEA and I = 1.20N + 0.59 (R2 = 0.9997) for a mixture. Both detection limits for two biomarkers were estimated to be 0.625 ng mL−1 . The precisions (2.5, 10 and 40 ng mL−1 ) were found to be 2.23–5.13% for CEA and 0.09–6.36% for NSE. In the presence of 40 ng mL−1 NSE, the recoveries of CEA (2.5, 10 and 40 ng mL−1 ) were 104.05, 100.03 and 99.29%, respectively. In the presence of 40 ng mL−1 CEA, the recoveries for NSE were calculated to be 90.86, 99.11 and 105.02%. Overall, our new technique allowed detection of two lung cancer markers down to subnanogram per milliliter level and was competitive with or even better than other assay formats (Table 1).
Table 1 Comparison of different multiplexed immunoassays. Analytical methods
Number of analytes
Label
Detection of limit
FL microarray Electrochemical immunoassay (ECL) Fluorescent immunoassay (FL) CL immunoassay CL immunoassay SERS Capacitive immunosensor CL immunoassay FL immunoassay CL immunoassay FL immunoassay FL immunoassay FL immunoassay ECL immunoassay Colorimetric immunoassay ICP-MS immunoassay ICP-MS immunoassay ECL immunoassay FL immunoassaya
6 4 4 4 4 3 3 3 3 2 2 2 2 2 2 2 2 2 2
FITC Colloidal nanocrystals QDs ALP ALP and HRP Silver nanoparticles Label-free ALP Alexa Fluor HRP QDs QDs QDs QDs HRP Eu3+ and Sm3+ Eu3+ and gold nanoparticles Gold nanoparticle QDs
20 ng mL−1 or 40 ng mL−1 [22] 10 ng mL−1 [10] – [27] 0.52 g L−1 , 0.49 kU L−1 , 0.79 kU L−1 , and 0.55 g L−1 [12] 0.15, 0.80, and 2.0 U mL−1 and 0.65 ng mL−1 [15] 100 pM [17] 25 pg mL−1 [34] 0.60 g L−1 , 0.080 g L−1 , and 0.70 kU L−1 [13] 0.2, 1 and 2 g mL−1 [35] 0.41 and 0.39 ng mL−1 [11] 1.3 and 1.2 ng mL−1 [31] 2.5 pM [32] 1 pM [29] 30 and 10 pg mL−1 [36] 0.02 ng mL−1 [37] 1.2 and 1.7 ng mL−1 [38] 2ng mL−1 [39] 1.0 and 1.5 ng mL−1 [40] 0.625 ng mL−1 (500 pM)
a
This work.
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Fig. 6. FL intensity of CEA and NSE in PBSTW-2% BSA medium containing different concentrations of NIS and FBS. Experimental conditions: CEA detection, 78 g PSM, 100 g mL−1 of NSE capture antibody, 40 g mL−1 of CEA detection antibody, and 1:600 of SA-QD; NSE detection, 78 g PSM, 60 g mL−1 of NSE capture antibody, 10 g mL−1 of NSE detection antibody, and 1:400 of anti-FITC-QD. The detection procedure was carried out as described in the Section 2.5.
Fig. 7. Comparison of CEA (A) and NSE (B) determinations in patient serum samples with the proposed method and commercial ELISA kits.
3.5. Application of the fluoroimmunoassay in human serum samples As shown in Fig. 6, FL intensity decreased with the increase of NIS, which was attributed to interference from NIS. Thus, different concentrations of FBS were investigated in parallel, and FL intensity was almost the same in the PBSTW-2% BSA medium containing equal percent of NIS or FBS (data of 30% FBS and NIS not shown). Therefore, PBSTW-2% BSA-50% FBS was employed to dilute and prepare the standard solutions for the construction of calibration curves and the analysis of real samples. The recoveries for 5, 20 and 80 ng mL−1 from 20 normal serum samples were calculated to be 95.01 ± 15.83%, 90.25 ± 18.61% and 94.63 ± 12.09% for CEA, respectively and 96.78 ± 16.53%, 102.30 ± 14.08% and 92.56 ± 6.40% for NSE, respectively. The assay results of clinical serum samples using the proposed method were compared with referred values obtained by commercial ELISA kits (data from Zhongshan Hospital). The results were shown in Fig. 7. Compared with the referred values, the good correlation was obtained (R2 > 0.90), indicating acceptable accuracy. 4. Conclusions A new duplex fluoroimmunoassay platform for the homogeneous determination of two biomarkers has been demonstrated. The optimal assay condition provides a good sensitivity to meet the requirement of cut-off levels and good reaction media for detection of clinical serum samples. Moreover, this new approach offers the following advantages: (1) two biomarkers can be detected in a single experiment with a single sample, consuming less time and sample. (2) Unlike the conventional biomarker detection assay, such as ELISA and heterogeneous fluoroimmunoassay, our new method employs PSM as the capture antibodies conjugated carrier to make the entire reaction occur in an almost homogeneous situation. (3) The wash procedure has been simplified by using a 96-well filter plate coupling with the vacuum filter device. Each wash can be finished within 20 s. (4) The FL signal derives from two cross-talk-free QD conjugated probes and easily obtained by
a prevalent fluorescent spectrometry. The whole 96-well plate can be scanned in 3 min, contributing to the practicality of its application. In summary, this QD-based immunoassay system has great potential to serve as the sensitive and specific cancer diagnostic tool in clinical test by virtue of its simplicity and efficiency. This new approach will have broad application in other biomarkers relating to other cancers, such as alpha fetoprotein (AFP) and prostate specific antigen (PSA) associated with liver cancer and prostate cancer, etc. Acknowledgements We acknowledge financial support from National 863 Program (2007AA03Z357), National Drug Innovative Program (2009ZX09301-011), the Research Fund for the Doctoral Program of Higher Education (200802461096) and National Basic Research Program of China (2007CB935800). We also thank Dr. Robert Polance for critical comments concerning this manuscript. References [1] A. Jemal, R. Siegel, E. Ward, Y. Hao, J. Xu, T. Murray, M.J. Thun, Cancer statistics, 2008, Can.-Cancer J. Clin. 59 (2009) 225–249. [2] H.J. Lee, A.W. Wark, R.M. Corn, Analyst 133 (2008) 975–983. [3] J.L. Gong, Y. Liang, Y. Huang, J.W. Chen, J.H. Jiang, G.L. Shen, R.Q. Yu, Biosens. Bioelectron. 22 (2007) 1501–1507. [4] S. Prabhulkar, S. Alwarappan, G. Liu, C.Z. Li, Biosens. Bioelectron. 24 (2009) 3524–3530. [5] J. Ladd, H. Lu, A.D. Taylor, V. Goodell, M.L. Disis, S. Jiang, Colloid Surf. B: Biointerfaces 70 (2009) 1–6. [6] H. Zhu, P.S. Dale, C.W. Caldwell, X. Fan, Anal. Chem. 81 (2009) 9858–9865. [7] A. Ambrosi, F. Airo, A. Merkoc, Anal. Chem. 82 (2010) 1151–1156. [8] X.A. Liu, Y.Y. Zhang, J.P. Lei, Y.D. Xue, L.X. Cheng, H.X. Ju, Anal. Chem. 82 (2010) 7351–7356. [9] R. Wilson, A.R. Cossins, D.G. Spiller, Angew. Chem. Int. Ed. 45 (2006) 6104–6117. [10] G. Liu, J.H. Wang, J. Kim, Anal. Chem. 76 (2004) 7126–7130. [11] Z.J. Yang, C. Zong, F. Yan, H.X. Ju, Talanta 82 (2010) 1462–1467. [12] H. Liu, Z.F. Fu, Z.J. Yang, F. Yan, H.X. Ju, Anal. Chem. 80 (2008) 5654–5659. [13] Z.F. Fu, F. Yan, H. Liu, J.H. Lin, H.X. Ju, Biosens. Bioelectron. 23 (2008) 1422–1428. [14] Z.F. Fu, F. Yan, H. Liu, Z.J. Yang, H.X. Ju, Biosens. Bioelectron. 23 (2008) 1063–1069. [15] Z. Fu, Z. Yang, J. Tang, H. Liu, F. Yan, H. Ju, Anal. Chem. 79 (2007) 7376–7382. [16] Z. Fu, H. Liu, H. Ju, Anal. Chem. 78 (2006) 6999–7005.
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