Magnetic Bead-Based Reverse Colorimetric Immunoassay Strategy

Article pubs.acs.org/ac

Magnetic Bead-Based Reverse Colorimetric Immunoassay Strategy for Sensing Biomolecules Zhuangqiang Gao, Mingdi Xu, Li Hou, Guonan Chen, and Dianping Tang* Key Laboratory of Analysis and Detection for Food Safety (Fujian Province & Ministry of Education of China), Department of Chemistry, Fuzhou University, Fuzhou 350108, P. R. China ABSTRACT: A novel reverse colorimetric immunoassay (RCIA) strategy was for the first time designed and utilized for sensitive detection of low-abundance protein (prostatespecific antigen, PSA, used in this case) in biological fluids by coupling highly catalytic efficient catalase with magnetic beadbased peroxidase mimics. To construct such a RCIA system, two nanostructures including magnetic beads and gold nanoparticles were first synthesized and functionalized with anti-PSA capture antibody and catalase/anti-PSA detection antibody, respectively. Thereafter, a specific sandwich-type immunoassay format was employed for determination of PSA by using functional gold nanoparticles as enzymatic bioreactors and anti-PSA-conjugated magnetic beads as a colorimetric developer. The carried catalase, followed by the sandwiched immunocomplex, partially consumed the added hydrogen peroxide in the detection solution, which slowed down the catalytic efficiency of magnetic bead-based peroxidase mimics toward TMB/ H2O2, thereby weakening the visible color and decreasing the colorimetric density. Different from conventional colorimetric immunoassay, the RCIA method determined the residual hydrogen peroxide in the substrate after consumption. Under the optimal conditions, the developed RCIA exhibited a wide dynamic range of 0.05−20 ng mL−1 toward PSA with a detection limit of 0.03 ng mL−1 at the 3Sblank level. Intra- and interassay coefficients of variation were below 6.1% and 9.3%, respectively. Additionally, the methodology was further validated for the analysis of 12 PSA clinical serum specimens, giving results in good accordance with those obtained by the commercially available enzyme-linked immunosorbent assay (ELISA) method.

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Perlmann in 1971, enzyme-mediated colorimetric immunoassays have passed through several dozens years development and have become an effective and indispensable instrument for medical laboratories, manufacturers of in vitro diagnostic products, regulatory bodies, and external quality assessment and proficiency-testing organizations.10,11 Recent research has looked to develop innovative and powerful labels based on enzyme-functionalized nanostructures to further achieve signal amplification. Ambrosi et al. designed a new enzyme-linked immunosorbent assay (ELISA) for the analysis of CA 15-3 antigen using gold nanoparticles as carriers of HRP-anti-CA153 to achieve the amplification of the optical signal.12 Zhang and co-worker developed a nanotube-based colorimetric probe using multiwalled carbon nanotubes and fabricated a colorimetric immunoassay for ultrasensitive detection of ataxia telangiectasia mutated protein.13 Due to the amplification of enzyme-functionalized nanostructures, the immunoassays could achieve an enhancement of the colorimetric signal. However, most of these methods were based on commonly used enzymes, e.g., HRP and ALP, and the colorimetric detection principle depended on measuring the direct color changes produced by the catalytic reaction of enzymes toward

ecently, colorimetric immunoassay has gained great attention in the various research areas, e.g., biomedical diagnosis, food safety analysis, and environmental monitoring, due to several important advantages, such as simplicity, practicality, low cost, and rapid/direct readout with the naked eye.1−7 A key challenge for the development of colorimetric immunoassay is to transform the detection event into color change.1 Various methods and strategies have been developed for this purpose, such as aggregation-based colorimetric immunoassay, lateral-flow colorimetric immunoassay, and enzyme-mediated colorimetric immunoassay. Aggregationbased colorimetric immunoassay and colorimetric lateral-flow immunoassay are user-friendly, fast, and cost-effective, which are usually employed for fast on-site analysis.6,8 Unfortunately, they often exhibited low sensitivity, because no signal amplification procedure was applied. Hence, the greatest attention has been recently focused on enzyme-mediated signal amplification methodologies, e.g., by employing enzymes and enzyme mimics as labels, especially for the determination of low-abundance proteins. Enzymes including horseradish peroxidase (HRP) and alkaline phosphatase (ALP) have shown great application values in the colorimetric immunoassays owing to their unique merits: high catalytic activity, high specificity, mild reaction conditions, and easy conjugation to antibodies and nanomaterials.5,9 Since the first one developed by Engvall and © XXXX American Chemical Society

Received: May 13, 2013 Accepted: June 14, 2013

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dx.doi.org/10.1021/ac401433p | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

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Scheme 1. Magnetocontrolled Enzyme-Mediated Reverse Colorimetric Immunoassay Protocol: (a) Design of Monoclonal Mouse Anti-Human PSA-Conjugated Magnetic Bead (MB-Ab1) and Polyclonal Goat Anti-Human PSA/Catalase-Labeled AuNP (multi-CAT-AuNP-Ab2), (b) Magnetocontrolled Enzyme-Mediated Reverse Colorimetric Immunosensing Strategy, and (c) Conventional Colorimetric Enzyme-Linked Immunosorbent Assay (ELISA)

substrates. Despite many advances in this field, there is still the quest for new schemes and protocols for improvement of simplicity and sensitivity of enzyme-mediated colorimetric immunoassays. For the successful development of enzyme-mediated colorimetric immunoassays, an assay platform is an important part. A reverse assay is a novel assay platform, whereby the absence of target triggers a change in the signal and the relationship between target concentration and signal change is inverse, and has attracted great interest in recent years owing to promising applications in various areas.14 Because some powerful detection labels and tags cannot generate a signal that is directly proportional to the concentration of the target molecule, reverse assays are developed to meet these challenges. Reverse assays have been used in a variety of bioassays. Liu and Lu developed a reverse colorimetric detection platform for a lead sensor based on the assembly of gold nanoparticles by a Pb2+-dependent DNAzyme.15 Stevens and co-workers reported a reverse plasmonic nanosensor with inverse sensitivity for detection of prostate-specific antigen by inducing a signal that is larger when the target molecule is less concentrated.16 Another important issue is the choice of bioactive enzymes. Catalase (CAT) has shown great potential in biosensing because it is more efficient and much less expensive than other popular alternatives such as HRP and ALP.17,18 The most outstanding characteristics of CAT is its ultrahigh catalytic property. It has one of the highest turnover numbers of all enzymes: one catalase molecule can convert millions of molecules of hydrogen peroxide (H2O2) to water and oxygen each second.19 Such a high catalytic property of CAT toward H2O2 is conducive for the construction of reverse colorimetric immunoassays. Owing to the advantages of the reverse assay model and catalase, our motivation in this work is to combine the ultrahigh catalytic activity of CAT with the merits of the reverse assay model to exploit a novel signal

generation method for developing a reverse colorimetric immunoassay with sensitivity enhancement. A gold nanoparticle label is an ideal candidate in the biotechnological systems due to its inherent advantages, such as easy preparation and good biocompatibility.20,21 Fe 3 O 4 magnetic beads (MBs) are efficient as peroxidase mimics and can enable convenient separation by simply applying an external magnetic field.22,23 Herein, we design a novel magnetocontrolled reverse colorimetric immunoassay for ultrasensitive detection of prostate-specific antigen (PSA, as a model protein) in biological fluids by using multi-CATfunctionalized gold nanoparticles as nanolabels and coupling the catalytic activity of CAT with the peroxidase-like catalytic activity of MBs to produce a colorimetric signal (Scheme 1). The assay mainly consists of the formation of a sandwiched immunocomplex, the CAT-mediated catalytic reaction, and the MB-mediated catalytic reaction. The aim of this work is to explore a novel reverse colorimetric immunoassay with sensitivity enhancement using multi-CAT-functionalized gold nanoparticles as an alternative to commonly used enzymes.

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EXPERIMENTAL SECTION Materials and Reagents. Mouse monoclonal anti-human PSA antibody (ab130880, designated as Ab1) was purchased from Abcam Inc. (Cambridge, MA). Goat polyclonal antihuman PSA antibody (designated as Ab2) was purchased from ImmunoReagents, Inc. (Raleigh, NC). PSA standards were obtained from Biocell Biotechnol. Co., Ltd. (Zhengzhou, China). Bovine liver catalase (CAT) and 3-aminopropyltriethoxysilane (APTES, 98%) were purchased from SigmaAldrich. 3,3′,5,5′-Tetramethylbenzidine (TMB) and bovine serum albumin (BSA) were purchased from Sinopharm Chem. Re. Co., Ltd. (Shanghai, China). Hydrogen peroxide (30 wt %, H2O2), ferric chloride hexahydrate (FeCl3·6H2O, >99%), ferrous chloride tetrahydrate (FeCl2·4H2O, >99%), anhydrous ethanol, ammonium hydroxide (NH4OH, 28 wt %), and B



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Figure 1. (A) UV−vis absorption spectra of (a) pure gold colloids and (b) multi-CAT-AuNP-Ab2. (B) Photographs of the mixture containing MBAb1 and multi-CAT-AuNP-Ab2 in the presence of (a) 0 and (b) 20 ng mL−1 PSA with an external magnet, respectively. (C, D) Typical TEM images of the mixture containing MB-Ab1 and multi-CAT-AuNP-Ab2 after incubation with (C) 0 and (D) 20 ng mL−1 PSA, respectively.

of PBS solution (containing 300 μL, 25% glutaraldehyde) at pH 7.4 and stirred for 6 h. After magnetic separation, the glutaraldehyde-functionalized MB (glu-MB) was dispersed in 1 mL of PBS (pH 7.4). Then 50 μL of the glu-MB solution was added to 500 μL of carbonate buffer (pH 9.6) containing 60 μg of Ab1 and shaken overnight at 4 °C. Following that, 10 μL of 10 wt % BSA in PBS was injected into the suspension and incubated for 2 h at 4 °C to block the possible residual sites on the MB. To reduce the resultant Schiff bases and any excess aldehydes, 50 μL of 25 mg mL−1 sodium cyanoborohydride was added to the suspension and incubated for 1 h at 4 °C. Afterward, the mixture was collected by applying an external magnetic field. The obtained pellet was resuspended into 0.5 mL of PBS (pH 7.4) containing 1.0 wt % BSA and 0.1 wt % NaN3 and stored at 4 °C for further usage. Preparation of Nanogold-Labeled CAT and Ab 2 Antibody (multi-CAT-AuNP-Ab2). Gold nanoparticles heavily functionalized with CAT and Ab2 antibody were prepared according to our recently reported methods.21,26 Briefly, 1.0 mL of 16-nm nanogold colloids was initially adjusted to pH 8−9 using 0.01 M NaOH, and then 20 μL of 100 μg mL−1 Ab2 and 80 μL of 100 μg mL−1 CAT were simultaneously added to the colloids. The resulting mixture was incubated overnight at 4 °C under gentle stirring. Afterward, the mixture was centrifuged (6500g) for 20 min at 4 °C to remove the excess proteins. The obtained pellet was resuspended into 1.0 mL of PBS (pH 7.4) containing 1.0 wt % BSA and stored at 4 °C for further usage. Immunoassay Protocol and Reverse Colorimetric Measurement. The reverse colorimetric immunoassay was carried out as follows: a 50-μL aliquot of PSA standards or samples with various concentrations was initially injected into a

glutaraldehyde (GA, 25 wt %) were obtained from Fuchen Chemicals (Tianjin, China). Gold colloids (AuNP) 16 nm in diameter were prepared and characterized as described.24 All other reagents were of analytical grade and were used without further purification. Ultrapure water obtained from a Millipore water purification system (18 MΩ, Milli-Q, Millipore) was used in all runs. A pH 9.6 carbonate buffer (1.59 g of Na2CO3, 2.93 g of NaHCO3, and 0.2 g of NaN3) and a pH 7.4 phosphatebuffered saline (PBS, 0.01 M) (2.9 g Na2HPO4·12H2O, 0.24 g of KH2PO4, 0.2 g of KCl, and 8.0 g of NaCl) were prepared by adding the corresponding chemicals into 1000 mL of distilled water, respectively. Preparation of Ab1-Conjugated Magnetic Bead (MBAb1). MB was synthesized according to the literature with slight modification.25 FeCl3·6H2O (0.5 g, 1.85 mmol) and FeCl2·4H2O (0.184 g, 0.925 mmol) were dissolved in deoxygenated distilled water (30 mL) that had been degassed by bubbling with N2 for 20 min. To this solution was added 7.5 mL of NH4OH (∼28% in water) under N2 atmosphere while stirring vigorously for 30 min. The resulting black precipitate was collected with the help of a magnet and thoroughly washed several times with deionized water. Then the black precipitate was washed three times with anhydrous ethanol and dried under vacuum at 40 °C for 12 h. The antibody-conjugated MB was prepared as described in our previous report.26 A 30 mg amount of dried MB was dispersed in 1 mL of anhydrous ethanol by ultrasonication for 2 h, 20 μL of APTES was added with stirring, and the solution was stirred continuously at room temperature for 6 h. Aminated MB was formed and separated by magnetic decantation. Subsequently, the resultant particles were redispersed in 1 mL C



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Figure 2. The effects of (a) sulfuric acid concentration and (b) H2O2 concentration on the peroxidase-like activity of fixed-amount magnetic beads. (c) The density of fixed-amount magnetic beads with various-concentration H2O2 from 0.1 to 0.01 mM relative to the H2O2−TMB system [Note: (d) The corresponding photographs].

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RESULTS AND DISCUSSION Construction and Characteristics of Reverse Colorimetric Immunoassay. Scheme 1 gives the fabrication process of the reverse colorimetric immunoassay (RCIA). Ab1 capture antibody was conjugated to the aminated MB through the cross-linking glutaraldehyde. The immobilization method could be easily implemented, as described in previous similar reports27,28 and characterized in detail in our previous papers.26,29 The association of CAT and Ab2 detection antibody to the colloidal surface was possibly due to the interaction between cysteine or NH3+-lysine residues of protein and gold nanoparticles.30 The successful formation of multi-CAT-AuNPAb2 conjugates were qualitatively characterized using UV−vis absorption spectroscopy. As shown in Figure 1A, the spectrum of bare AuNP (curve a) exhibited a characteristic plasmon absorption peak at 517 nm. After the interaction between AuNP and CAT/Ab2, the plasmon absorption peak shifted from 517 to 520 nm (curve b), indicating the formation of bioconjugates.31,32 Scheme 1b illustrates the assay principle of the RCIA toward target PSA. In the presence of target PSA, the sandwiched immunocomplex was formed between MB-Ab1 and multi-CATAuNP-Ab2. The carried CAT could catalyze the reduction of H2O2 in the detection solution, and consumed the partial H2O2, thereby slowing down the catalytic efficiency of MB toward TMB/H2O2. As such, the absorbance was decreased and the visible color was weakened. By monitoring the decrease in the absorbance, we could quantitatively determine the concentration of target PSA in the sample. Meanwhile, we could also qualitatively determine the PSA level by the change in visible color. In contrast, in the absence of target PSA, the sandwiched immunocomplex could not be formed. To verify

0.5-mL centrifuge tube, and a 25-μL aliquot of MB-Ab1 suspension (3 mg mL−1) was then introduced into the centrifuge tube. After incubation for 30 min at 37 °C on an end-over-end shaker (MS, IKA GmbH, Staufen, Germany), the resulting mixture was separated simply with an external magnet and washed with PBS. Afterward, a 50-μL aliquot of the aboveprepared multi-CAT-AuNP-Ab2 was added into the centrifuge tube and incubated for an additional 30 min under the same condition. The formed immunocomplex between MB-Ab1 and multi-CAT-AuNP-Ab2 was separated and washed as the above protocol. Following that, 50 μL of 0.1 mM H2O2 in PBS was added into the tube and shaken for 30 min on an end-over-end shaker for catalysis. Subsequently, 20 μL of 2 M H2SO4 and 50 μL of 0.921 mM TMB were added to the tube and shaken for 30 min for color development. Meanwhile, the absorbance was read at 450 nm with a plate reader (DNM-9602, Beijing Perlong Medical Instrument Ltd., China). All measurements were conducted at room temperature. Analyses are always made in triplicate. Enzyme-Linked Immunosorbent Assay (ELISA) for PSA. A commercially available ELISA kit was utilized for method comparison. In the sandwich ELISA with standard polystyrene 96-well plates, 50 μL of serum sample suspension was incubated at 37 °C for 60 min, and the wells were rinsed three times (3 min each) with 0.1 M PBS (pH 7.4) containing 0.05% (v/v) Tween 20. Then we added 50 μL of anti-PSAHRP conjugate solution, and incubation continued for 60 min. After washing, TMB reagent (50 μL) and H2O2 (50 μL) was added and incubated at 37 °C for 10 min. The enzymatic reaction was stopped by adding 50 μL of 2.0 M H2SO4 to each well. The results of ELISA were measured by a spectrophotometric ELISA reader at a wavelength of 450 nm. D



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Figure 3. Comparison of several control tests: (A) the absorbance densities of (a) MB + TMB + H2SO4 + H2O, (b) MB + TMB + H2SO4 + H2O2, (c) H2O2 + CAT + H2SO4 + MB + TMB, and (d) H2O2 + CAT + TMB + H2SO4, respectively (Note: The corresponding photographs); (B) the absorbance densities of (a) MB + TMB + H2SO4 + H2O2 and (b) MB + TMB + H2SO4 toward the CAT with various concentrations, respectively.

Within the working range, the absorbance decreased with the decreasing H2O2 concentration. To acquire a wide linear range and low detection limit of the developed RCIA, 0.1 mM H2O2 was employed for the development of visible color in the following RCIA. Control Test. To realize our design, several control tests were carried out under the same experimental conditions (Note: 5 μL of 15 mg mL−1 glu-MB, 20 μL of 2 M H2SO4, 50 μL of 0.921 mM TMB, 50 μL of 0.1 mM H2O2 in pH 7.4 PBS, and 5 μL of 1 μg mL−1 CAT were used in this case, respectively. Incubation: 30 min at 37 °C). As shown from column a in Figure 3A, a very weak absorbance was observed toward the mixture containing glu-MB, TMB, and H2SO4. When H2O2 was added into the mixture, however, a strong absorbance (A450 nm = 0.942) was acquired (column b in Figure 3A). Meanwhile, the visible color was changed from colorless (photograph a in Figure 3A) to yellow (photograph b). The result indicated that (i) MB with the peroxidase-like activity could not directly catalyze the TMB in the absence of H2O2, and (ii) use of sulfuric acid could not stop the MB-based catalytic reaction relative to the TMB−H2O2 system. The reason might be the fact that MB-based peroxidase mimics were a kind of inorganic nanomaterial and could not be readily denatured under the strong acidic condition (Note: The bioactivity of HRP, as a kind of protein, was easily denatured at the low pH). Hence, the presence of H2O2 was a precondition for the development of visible color during the RCIA measurment. In this work, the assay was based on monitoring the residual amount of the fixed-concentration H2O2 in the detection solution after the formation of the sandwiched immunocomplex. The consumption of H2O2 mainly derived from the labeled CAT on the AuNP. To verify this issue, 50 μL of 0.1 mM H2O2 in pH 7.4 PBS and 5 μL of 1 μg mL−1 CAT (as an example) were initially incubated for 30 min at 37 °C, and then the MB/TMB/H2SO4 mixture was added into the resultant solution, which was incubated for 30 min under the same condition. As seen from column c in Figure 3A, the absorbance was largely decreased (A450 nm = 0.398), indicating that the added CAT partially consumed the present H2O2, thus resulting in the decrease of the absorbance and the weakness of visible color (photograph c in Figure 3A). Logically, another question arises as to whether CAT, as an extraordinary enzyme in reproductive reactions, could catalyze the TMB in the presence of H2O2. As indicated from column d in Figure 3A, the absorbance was not nearly changed when CAT, TMB, and

this issue, TEM images of MB-Ab1 and multi-CAT-AuNP-Ab2 were investigated (Figure 1C) before and (Figure 1D) after addition of target PSA to the resultant mixture. As seen from Figure 1C, most of nanoparticles were homogeneously dispersed in the solution. In the presence of target PSA, many nanoparticles were aggregated together (Figure 1D). Faintly, we also observed that many gold nanoparticles (pale dots) were attached around the MBs (black dots). As indicated from Figure 1B, the color of the supernatant by magnetic separation was obviously lighter in the presence of target PSA than that of the absence of target PSA. The results revealed that most of the gold nanoparticles were conjugated onto the MBs by target PSA. Optimization of Experimental Conditions. To achieve an optimal analytical performance, the experimental conditions including the concentrations of H2SO4 and H2O2 for catalytic reaction of MB toward TMB/H2O2 should be examined. It has been proved that the peroxidase-like activity of MB is much higher in acidic solution than in neutral or basic solutions.33 The reasons were most likely a consequence of the facts that (i) Fenton’s reagent (i.e., Fe2+/Fe3+ ions in solution) can catalyze the breakdown of H2O2, and the iron ions contained in the MB might leach into the reaction buffer solution, and (ii) Fe2+ ions in the MB may play a dominant role in the catalytic peroxidaselike activity.22,33 Figure 2a shows the peroxidase-like activity of the synthesized glu-MB toward sulfuric acid with various concentrations in the presence of 5 μL of 15 mg mL−1 glu-MB, 20 μL of H2SO4, 50 μL of 0.921 mM TMB, and 50 μL of 0.1 mM H2O2. As seen from Figure 2a, the absorbance increased with the increasing sulfuric acid concentration and tended to level off after 2 M. Hence, a sulfuric acid concentration of 2 M was chosen for catalytic reaction of glu-MB toward TMB and H2O2 in the RCIA. In the developed RCIA, the concentration of H2O2 was a very import factor influencing the sensitivity of the immunoassay. In this case, 5 μL of 15 mg mL−1 glu-MB, 20 μL of 2 M H2SO4, and 50 μL of 0.921 mM TMB were used for the detection of 50 μL of H2O2 with various concentrations. As indicated from Figure 2b, the mixture exhibited a sigmoidal response curve toward various-concentration H2O2. The leaping range in the absorbance was 0.01−0.1 mM H2O2. When the concentration of H2O2 was higher/lower than 0.1 mM/0.01 mM, the change in the absorbance was not obvious. Moreover, it displayed a good linear relationship within the dynamic range of 0.01−0.1 mM toward H2O2 (A = 8.128 × c(H2O2) (mM) + 0.1133 (R2 = 0.997, n = 30)) (Figure 2c,d). E



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Figure 4. Calibration plots of various immunoassay protocols toward PSA standards with various concentrations using (a) the reverse colorimetric immunoassay strategy and (b) conventional colorimetric ELISA method, respectively (Insets: the corresponding photographs).

Figure 5. (a) The specificity of the reverse colorimetric immunoassay toward PSA (5 ng mL−1), AFP (50 ng mL−1), CEA (50 ng mL−1), CA 15−3 (50 U mL−1), CA 19−9 (50 U mL−1), CA 125 (50 U mL−1), and HIgG (50 ng mL−1). (b) The corresponding photographs for Figure 5a.

= cCAT/12 = 2.083 × 10−17 mol/12 = 1.736 × 10−18 mol). With this correspondence, the concentration of PSA is about 1.146 pg mL−1 in 50-μL detection solution in principle (cPSA = nPSA × MPSA/VPSA = 1.736 × 10−18 mol ×33 000 g mol−1/0.05 mL = 1.146 × 10−12 g mL−1 = 1.146 pg mL−1, molecular weigh of PSA is 33 kDa). The result provided a theoretical basis for ultrasensitive detection of target PSA. As a control test, glu-MB + TMB + H2SO4 were also used for the detection of CAT in the absence of H2O2 (curve b in Figure 3B). The results, along with the absence of signal change in the experiment in the present of H2O2 but in the absence of CAT, demonstrated that the decomposition reaction of H2O2 by the biocatalytic activity of the CAT enzyme is the key factor for the changes of the absorbance in glu-MB/H2O2/TMB system, and the signalgeneration mechanism of the developed RCIA is based on the biocatalytic activity of the CAT toward H2O2, as depicted in Scheme 1b. Analytical Performance of Reverse Colorimetric Immunoassay. Under optimal conditions, the sensitivity and quantitative range of the RCIA were studied by assaying routine samples with different PSA concentrations based on the developed protocol. The absorbance density decreased with the increasing PSA concentration in the sample (Figure 4a). A linear dependence between the absorbance and the logarithm of PSA level was obtained in the range from 0.05 ng mL−1 to 20 ng mL−1. The detection limit (LOD) was 0.03 ng mL−1 estimated at a signal-to-noise ration of 3 (n = 15). Because the threshold of total PSA in normal humans is about 4 ng mL−1, the developed RCIA can completely meet the requirement of clinical diagnostics. For comparison, we also

H2O2 were reacted for 30 min at 37 °C followed by H2SO4. The result suggested that CAT was completely different from the classical horseradish peroxidase (HRP), which could not directly be used in the ELISA format. This is also one of the important reasons why we used the RCIA. To further clarify the feasibility of the developed RCIA, two types of assay protocols with and without H2O2 (i.e., MB + TMB + H2SO4 + H2O2 and MB + TMB + H2SO4) were used for the detection of CAT with various concentrations, respectively (Note: The added sequence was the same as the above-mentioned mode). As seen from curve a in Figure 3B, the absorbance decreased with the increasing CAT concentration in the presence of H2O2. The linear range of using 5 μL of 15 mg mL−1 glu-MB, 20 μL of 2 M H2SO4, 50 μL of 0.921 mM TMB, and 50 μL of 0.1 mM H2O2 as the substrates was 1.0−10000 ng mL−1 toward CAT. That is to say, only if the concentration of CAT was higher than 1.0 ng mL−1 was the absorbance obviously changed. Herein, we might roughly estimate that one 16-nm gold nanoparticle could simultaneously conjugate twelve CAT molecules at most (Note: The calculation is based on the assumption of spherical surface area [SNP = 4πrNP2] divided by the area of the catalase’s radius-based circle [SCAT = πrB2], where rNP stands for the radius of AuNP, rB stands for the radius of CAT, and the hydrodynamic diameter of catalase is around 9 nm).34,35 Because 5 μL of 1 ng mL−1 CAT containing 2.083 × 10−17 mol CAT could lead to a decrease in the absorbance (nCAT = cCAT × VCAT = 1 × 10−9 g mL−1/250 000 g mol−1 × (5 × 10−6)L = 2.083 × 10−17 mol, molecular weigh of CAT is 250 kDa), the detectable amount of multi-CAT-AuNP-Ab2 is 1.736 × 10−18 mol (nmulti‑CAT‑AuNP‑Ab2 F



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investigated the analytical properties of the conventional colorimetric ELISA kit (Biocell Biotechnol. Co., Ltd., Zhengzhou, China) strategy based on HRP-labeled Ab2 (Figure 4b). The linear range and LOD were 5−50 ng mL−1 and 0.5 ng mL−1 PSA, respectively. Although the system has not yet been optimized for maximum efficiency, the LOD of the developed RCIA was close to 20-fold lower than that of conventional ELISA method. Significantly, the system was capable of continuously carrying out all steps in less than 2.5 h for one sample, including incubation, washing, enzymatic reaction, and measurement, which is almost the same as that of the commercialized ELSIA kit (∼3 h). The reproducibility and precision of the developed RCIA were monitored by assaying 0.05, 0.5, and 10 ng mL−1 PSA as examples, using identical batches of MB-Ab1 and multi-CATAuNP-Ab2. Experimental results indicated that the coefficients of variation (CVs, n = 6) of the intra-assay were 3.1%, 6.1%, and 5.9% for 0.05, 0.5, and 10 ng mL−1 PSA, respectively, whereas the CVs of the interassay with various batches were 9.3%, 7.8%, and 5.4% (n = 6) at the above-mentioned concentrations, respectively. Hence, both intra-assay and interassay verified acceptable reproducibility and further revealed the possibility of batch preparation. To evaluate the specificity of the developed RCIA for PSA detection, we challenged the system with other interfering substances in human serum, e.g., α-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen 15-3 (CA 15-3), cancer antigen 19-9 (CA 19-9), cancer antigen 125 (CA 125), and human immunoglobulin G (HIgG). The assay was implemented with the same experimental procedures. As shown from Figure 5, a significant change in the absorbance density was observed with the target PSA toward 10-fold higher interfering components. These results revealed that the components coexisting in the sample matrix did not interfere in the determination of PSA, i.e., the developed RCIA was shown to be sufficiently selective for the detection of target PSA. Analysis of Real Samples and Evaluation of Method Accuracy. To monitor the analytical reliability and applicable potential of the developed RCIA for testing real samples, we collected 12 human serum specimens with various PSA levels from Fujian Provincial Hospital of China according to the rules of the local ethical committee. Prior to measurement, these samples were gently shaken at RT (Note: all handling and processing were performed carefully, and all tools in contact with patient specimens and immunoreagents were disinfected after use) and then evaluated by using the developed RCIA. The assayed results were compared with those obtained by using the commercially available human PSA ELISA kit. The results are listed in Table 1. Statistical analysis of the experimental results was performed using a t-test and linear regression analysis between the two methods. As seen from Table 1, the texp values in all samples were less than tcrit (tcrit[4, 0.05] = 2.77). Moreover, the slope and intercept of the regression equation between two methods were close to the ideal values 1 and 0, respectively, thereby revealing a good agreement between both analytical methods.

Table 1. Comparison of the Assay Results for Human Serum Specimens by Using the Developed RCIA and the Commercially Available ELISA Method method;a concentration [mean ± SD (RSD, %), n = 3, ng mL−1] sample no.b

found by the developed RCIA ± ± ± ± ± ± ± ± ± ± ±

texp

found by ELISA

1 2 3 4 5 6 7 8 9 10 11

1.54 2.61 3.36 4.65 2.03 2.27 1.17 9.45 16.7 8.77 0.375

0.20 0.21 0.31 0.64 0.21 0.18 0.06 0.71 1.4 0.88 0.022

1.44 2.52 3.62 4.32 1.87 2.41 1.20 10.5 15.9 7.93 no application

12

0.087 ± 0.013

no application

± ± ± ± ± ± ± ± ± ±

0.11 0.08 0.03 0.09 0.06 0.10 0.08 0.9 0.6 0.27

0.79 0.68 1.34 0.88 1.27 1.20 0.57 1.65 0.95 1.58 no application no application

a

The regression equation (linear) for these data is as follows: y = 0.966x + 0.095 (R = 0.994, n = 30) (x-axis: by the reverse colorimetric immunoassay; y-axis: by ELISA). bSamples 1−7 were clinical serum specimens, and samples 8−12 were the diluted samples by using newborn cattle serum.

peroxidase mimic. Compared with the conventional colorimetric immunoassay system, the developed reverse colorimetric immunoassay adequately utilizes the peroxidase-like activity of magnetic beads and high efficiency of catalase toward catalytic reduction of hydrogen peroxide. Necessarily, one overwhelming concern on the developed RCIA is the selection of the concentration of hydrogen peroxide within the range of leaping points, because it directly determines the sensitivity of the reverse colorimetric immunoassay. Highlights of this work mainly focus on the following issues: (i) magnetic beads are not only utilized as the substrates for the conjugation of capture antibody but are also used as peroxidase-like mimics for the development of visible color relative to the TMB−H2O2 system; (ii) the developed RCIA method can pull antibodies bound to magnetic beads from one laminar flow path to another by applying a local magnetic filled gradient and selectively remove them from flowing biological fluids without any washing steps; (iii) the use of catalase with highly efficient catalytic activity is conducive for the development of the RCIA in the limited amount of hydrogen peroxide, because catalase has almost no ability to catalyze the TMB in the presence of H2O2. More inspiringly, the RCIA-based assay method does not require sophisticated instruments and is well suitable for high-throughput biomedical sensing and application in both clinical and biodefense areas by controlling the target antibody.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86-591-2286 6125. Fax: +86-591-2286 6135. E-mail: [email protected].

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Notes

The authors declare no competing financial interest.

CONCLUSIONS In summary, this work demonstrates the development of an advanced reverse colorimetric immunoassay for the detection of low-abundance protein in biological fluids with high selectivity and sensitivity by coupling with bioactive enzyme and

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ACKNOWLEDGMENTS This work was financially supported by the National “973” Basic Research Program of China (2010CB732403), the G



Article

(34) Chirra, H.; Sexton, T.; Biswal, D.; Hersh, L.; Hilt, J. Acta Biomater. 2011, 7, 2865−2862. (35) Zhang, J.; Chi, Q.; Zhang, B.; Dong, S.; Wang, E. Electroanalysis 1998, 10, 738−746.

National Natural Science Foundation of China (41176079, 21075019), the Doctoral Program of Higher Education of China (20103514120003), the National Science Foundation of Fujian Province (2011J06003), the China-Russia Bilateral Scientific Cooperation Research Program (NSFC/RFBR) (21211120157), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT1116).

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