Article pubs.acs.org/ac
Gold Nanorods as Colorful Chromogenic Substrates for Semiquantitative Detection of Nucleic Acids, Proteins, and Small Molecules with the Naked Eye Xiaoming Ma,†,∥ Zhitao Chen,†,‡,∥ Palanisamy Kannan,§ Zhenyu Lin,† Bin Qiu,† and Longhua Guo*,† †
Institute of Nanomedicine and Nanobiosensing, The Key Lab of Analysis and Detection Technology for Food Safety of the MOE and Fujian Province, College of Chemistry, Fuzhou University, Fuzhou, 350116, China ‡ Fuqing Entry-Exit Inspection & Quarantine Bureau of P. R. China, Fuqing, 350300, China § Singapore Centre on Environment Life Sciences Engineering, Nanyang Technological University, 60 Nanyang Drive, SBS-01N-27, 637457, Singapore S Supporting Information *
ABSTRACT: Herein, we report for the first time a colorful chromogenic substrate, which displays vivid color responses in the presence of different concentration of analytes. Our investigation reveals that the selective shortening of gold nanorods (AuNRs) could generate a series of distinct colors that covers nearly the whole visible range from 400 to 760 nm. These vivid colors can be easily distinguished by the naked eye; as a result, the accuracy of visual inspection could be greatly improved. Next, we demonstrate the utility of AuNRs as multicolor chromogenic substrate to develop a number of colorimetric immunoassay methods, e.g., multicolor enzyme-linked immunosorbent assay (ELISA), multicolor competitive ELISA, and multicolor magnetic immunoassay (MIA). These methods allow us to visually quantify the concentration of a broad range of target molecules with the naked eye, and the obtained results are highly consistent with those state-of-the-art techniques that are tested by the sophisticated apparatus. These multicolor portable and cost-effective immunoassay approaches could be potentially useful for a number of applications, for example, in-home personal healthcare, on-site environmental monitoring, and food inspection in the field.
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Hitherto, many efforts have been paid to explore the diverse colorimetric sensors for biological and biomedical applications.11−14 Among these sensors, many of them can generate visual distinguishable color changes.15−21 However, the accuracy of visual inspection is poor due to the limited color display. As a result, naked-eye inspection is normally used for qualitative detection, while a spectrometer is still necessary to be used for quantitative determination of the concentration of the targets.15,22 The necessity of using an additional spectrometer not only increases the cost but also reduces the portability of the detection system. Therefore, the development of the colorimetric sensor that enables naked-eye semiquantitative detection of the targets could be greatly desired. As we all know, human eyes are insensitive to optical density variations (no spectral peak shift) but sensitive to color variations (with spectral peak shift).15 Experts estimated that as many as 10 million colors can be distinguished by human eyes.23 Therefore, it is predictable that the accuracy of visual inspection could be significantly improved, if a multiple colors
ortable and affordable devices for sensitive detection of a broad range of target molecules could be very attractive for a number of applications including clinical diagnosis, on-site environmental monitoring, and food inspection in the field.1−4 For example, in resource constrained countries and regions, sophisticated medical facilities may not be available, thus the development of affordable devices is highly important for medical diagnosis of various diseases, which greatly improves the health conditions of people’s living in such areas.5,6 Portable and affordable personal healthcare devices are also welcome for people living in urban areas in developed countries as well as rural areas in developing countries.7,8 Besides that, in many cases, the environmental monitoring and food safety inspection require on-site detection of the target molecules; hence, many routine methods conducted in the laboratory utilizing large equipment are not applicable.9,10 In this case, portable yet highly sensitive and selective devices could be good alternative candidates. In order to realize the portable and low-cost devices, the sensing elements of such devices should be as simple as possible. In this regard, a colorimetric sensor is considered as a potential candidate that enables naked-eye detection of the target molecules without using any special instruments. © 2016 American Chemical Society
Received: December 5, 2015 Accepted: February 11, 2016 Published: February 11, 2016 3227
DOI: 10.1021/acs.analchem.5b04621 Anal. Chem. 2016, 88, 3227−3234
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AuNRs solution was added into each tube. Next, 0.30 mL of H2O2 solution with different concentrations (0, 0.05, 0.1, 0.3, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 3.8, 4.2, and 5.0 mM, respectively) was added into the sequentially numbered centrifuge tubes (1− 14). Finally, 0.15 mL of 2 M HCl and 0.05 mL of 20 mM FeSO4 solutions were added into the above sequenced tubes to initiate the etching process. The solution was incubated at room temperature for 15 min. Then, the absorbance spectra of the solution were taken by a portable UV−vis spectrometer (Maya2000 Pro, Ocean Optics). The photographs of the solution were taken by a DSLR camera (Canon EOS 600D). Preparation of Antibody-Catalase and DNA-Catalase Conjugates. To prepare anti-AFP antibody-catalase conjugate, the anti-AFP antibody was dissolved in 1.0 mL of phosphate buffer saline (PBS, 10 mM, pH 7.4) to obtain a final concentration of 0.1 mM. Then 0.87 mg of SM (PEG)12 cross-linker (Thermo Scientific) was added. The reaction mixture was incubated at room temperature for 30 min. Then the excess cross-linker was removed by using a desalting column (Zeba Desalt Spin Columns, Thermo Scientific) equilibrated with PBS (10 mM, pH 7.4). Next, 1.0 mL of catalase solution (0.5 mM, dissolved in PBS) was added and then incubated at 4 °C for 2 h. Finally, the antibody-catalase conjugate was stored at 4 °C until use. The same procedures were used to prepare the DNA-catalase conjugate by replacing of anti-AFP antibody with aminemodified DNA probes. Preparation of Aflatoxin B1 (AFB1)-Catalase Conjugate. AFB1-catalase conjugate was synthesized according to the method described in the literature.29 The final AFB1catalase conjugate stock solution was ∼4.8 × 10−6 M, as calculated by the adsorption of catalase at 280 nm. The stock solution was stored at −20 °C for further use. The AFB1catalase working solution was prepared by diluting the stock solution 105 times with deionized water shortly before use. Preparation of Oligonucleotide-Magnetic Beads (MBs) Conjugate. The locked nucleic acid (LNA) modified DNA probe was attached onto the surface of MBs via streptavidin−biotin interaction. Briefly, 200 μL streptavidin functionalized magnetic beads (0.5% w/v) was washed with PBS (10 mM, pH 7.4) for three times and the supernatant was discarded. The MBs were then dispersed in 1 mL of PBS (10 mM, pH 7.4) containing biotinylated DNA probe (2 O.D). The solution was incubated at 37 °C for 30 min. Then, the DNA functionalized MBs were separated and washed with PBS (10 mM, pH 7.4) three times. The DNA functionalized MBs were blocked by the addition of 1 mL of PBS (10 mM, pH 7.4) containing 5% BSA at 37 °C for 60 min. Detection of Human Alpha Fetoprotein (AFP) via Conventional sandwich-Format ELISA. All reagents to conduct the ELISA were adopted from a commercial available Human AFP ELISA Kit (Alpha Diagnostic Intl. Inc.) except for AuNRs and anti-AFP antibody-catalase conjugate. Detail experimental procedures to conduct the ELISA please refer to the specification of the kit and can also be found in the Supporting Information. After enzymatical reaction, 75 μL of AuNRs, 22.5 μL of 2 M HCl, and 7.5 μL of 20 mM FeSO4 solutions were added into the solution to generate the colorimetric reaction. The absorbance spectra of the final solution was measurecd with a 96-well UV-STAR plate (Greiner). Also the representative photos of the solution were taken by a DSLR camera (Canon EOS 600D).
are presented in a colorimetric sensor. For example, the widerange pH test paper is the most well-known multicolor sensor that is able to quantify the hydrogen ion with the naked eye. The color of the pH test paper varied among red, orange, yellow, green, and violet in the presence of different concentration of hydrogen ions.24 These vivid colors highly improved the accuracy of visual inspection, so that people can identify the pH value without the use of a pH meter. Regretfully, the pH test paper can only sensing the concentration of hydrogen ions, thus multicolor sensors that can be used for visual quantification of a broad range of targets with the naked eye are still challenging. Colorimetric immunosensor is a potential alternative method because the target molecules of immunosensors could be broad due to the well-developed antigen−antibody immunoassay systems. However, most of the reported chromogenic substrates such as 3,3′,5,5′-tetramethylbenzidine (TMB),25 o-phenylenediamine (OPD),26 and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)27 show only a monochromic intensity change in the presence of the target molecules. These single color responses seriously confine the accuracy of visual inspection. Therefore, the development of highly sensitive colorful chromogenic substrate for colorimetric immunosensing that shows muticolor responses is very important in order to improve the accuracy of visual inspection. Thus, in this work we demonstrate a multicolor sensing strategy that can be used for visual quantification of a variety of analytes with the naked eye. The mechanism of the proposed sensor is based on target-molecule guided etching of gold nanorods (AuNRs). During the etching process, AuNRs are selectively shortened and eventually turned into the Au(III) state. The morphology change of AuNRs leads to a significant shift in the corresponding extinction spectrum. As a consequence, the color of the solution varied vividly in the presence of varied concentration of analyte molecules during this etching process. For example, brownish, gray, cyan, green, blue, violet, red, colorless, and yellow solutions are observed in response with different concentration of analytes. It is worth to note that these colors are even more abundant than the widely used pH test paper. Thus, the combination of the vivid color display of AuNRs and conventional immunoassay strategies enables us to visually quantify a variety of analytes ranging from proteins to nucleic acids and small molecules with the naked eye.
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EXPERIMENTAL SECTION Preparation and Characterization of AuNRs. An improved seed-mediated growth method using 5-bromosalicylic acid as an additive agent was adopted for the preparation of monodisperse AuNRs.28 The extinction spectra of AuNRs were measured by using portable Maya2000 Pro UV−vis spectrometer (Ocean Optics, Dunedin, FL) at room temperature. Transmission electron microscopy (TEM) images were acquired on a Tecnai G2 F20 S-TWIN microscope operating at 200 kV. Before TEM characterization, AuNRs were centrifuged at 10 000 rpm for 20 min. Then, the precipitates were redispersed into deionized water with the same volume and centrifuged again at 10 000 rpm for 10 min. The supernatant was discarded and the precipitate was dropped carefully onto a lacey-Formvar TEM grid and allowed to dry in air overnight before TEM analysis. Detection of H2O2. Centrifuge tubes (1.5 mL) were numbered sequentially, and then 0.5 mL of as-synthesized 3228
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Figure 1. NEQ assay for visual quantification of H2O2. (a, b) UV−vis spectra and corresponding photographs of the solutions in the presence of different concentrations of H2O2. The concentrations of H2O2 were 0, 0.05, 0.1, 0.3, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 3.8, 4.2, and 5.0 mM, respectively, corresponding to the sample number of 1 to 14; (c) representative TEM images of AuNRs during the etching process. The scale bars were representing 100 nm and the insets were the photographs of corresponding solutions used for the TEM analysis.
Detection of AFB1 via Competitive ELISA. AFB1 standard solution was diluted with deionized water to 0.02, 0.05, 0.08, 0.10, 0.12, 0.14, 0.16, 0.18, 0.20, 0.22, 0.24, 0.26, 0.28, 0.30, 0.32, 0.34, 0.36, 0.38, 0.40, and 0.42 ng/mL, respectively. Rice samples spiked with AFB1 were prepared as follows: Rice sample (2 g) was homogenized with 5 mL of 70% methanol solution and incubated at 37 °C for 5 min with constant shaking. The solution was then centrifuged at 4 000 rpm for 10 min; 120 μL of supernatant was taken out, and 120 μL of deionized water was added. After mixing, the extracts were divided into six aliquots (40 μL each). Finally, 10 μL of AFB1 standard solutions with the concentrations of 0.3, 0.6, 0.9, 1.2, 1.5, and 2.0 ng/mL were spiked into the respective extracts. These spiked samples were used to simulate AFB1 contaminated samples. All reagents to conduct the ELISA were adopted from a commercial available AFB1 ELISA Kit (MyBioSource, San Diego, CA) except for AuNRs and AFB1-catalase conjugate. Detail experimental procedures to conduct the competitive ELISA please refer to the specification of the kit and can also be found in the Supporting Information. Detection of miRNA via Magnetic Immunoassay (MIA). The sample or standard solution (500 μL) was added into separate centrifuge tubes (1.5 mL volume), and 20 μL of DNA probe coated MBs (0.1% w/v) was added to each tube. Then 500 μL of PBS (40 mM, pH 7.4) was added into the above tube. The mixture solution was incubated at 37 °C for 30 min. Then, the MBs were precipitated with a magnet and the supernatant was discharged. Next, 20 μL of DNA-catalase conjugate was added to the centrifugal tube, and then 100 μL of reaction buffer solution (5 mg/mL BSA, and 20 mM of PBS, pH 7.4) was added, and the solution was incubated at 37 °C for 30 min. Then, MBs were precipitated with a magnet and the supernatant was discharged. The MBs were washed with PBS (20 mM, pH 7.4) for three times. Finally, enzymatic reaction was generated by the addition of 300 μL of H2O2 solution (8.5 mM H2O2 in 0.1 M PBS) to the precipitate and incubated at room temperature for 60 min. The MBs were precipitated with a magnet and the supernatant was transferred into a 1.5 mL centrifuge tube. Then 0.5 mL of as-synthesized AuNRs solution was added into the tube. Next, 0.15 mL of 2 M HCl and 0.05 mL of 20 mM FeSO4 solution were added to catalyze the colorimetric reaction. The solution was incubated at room
temperature for 15 min. The representative photos of the solution were recorded with a DSLR camera (Canon EOS 600D). For the detection of miRNA-21 in HeLa cells, miRNA-21 was extracted with a miRNeasy Mini Kit (Qiagen) follow the kit handbook (detail procedures can be found in the Suporting Information). The final solution was collected for detection of miRNA-21 using the same steps for the analysis of standard solution. Procedures for Visual Quantification of the Analytes. In order to enhance the accuracy of visual quantification, standard solutions with narrow concentration differences were tested for each analyte, and the colors of the standard solutions were utilized as standard references for visual quantification of an unknown sample. Typically, visual quantification of the concentrations of targets in the unknown samples were accomplished as follows: The color of the sample solution was compared with those of the standard solutions. If the color of the sample was close to the color of one of the standard solutions, then the concentration of the standard solution was designated as the target concentration of the sample; if the color of the sample was in between the colors of two standard solutions, then the average values of the two standard solutions was designated as the target concentration of the sample.
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RESULTS AND DISCUSSION Feasibility of AuNRs for Visual Quantification of Target Molecules. Generally, sensors for naked-eye semiquantitative (NEQ) assay should at least satisfy two conditions: first, the sensor should exhibit vivid color variations so that visual distinguishing the different colors can be easily achieved; second, it should have one-to-one correspondence between the concentration of the analytes and the corresponding color display. Herein we investigated the feasibility of using AuNRs as multicolor chromogenic substrate for conducting NEQ assay. In order to testify the feasibility of AuNRs for NEQ assay, AuNRs were first demonstrated for semiquantitative determination of H2O2 with the naked eye. It has been reported that high concentration of H2O2 can be used as an oxidant for anisotropic shortening of AuNRs at room temperature.30 In the presence of a relatively high concentration of CTAB, the oxidation started at the ends of the AuNRs, thus the length of AuNR decreased, while the diameter was almost unchanged. 3229
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substances, which involve the production or consumption of H2O2. The rest of this work would show how to utilize AuNRs for visual quantification of proteins, DNAs, and small molecules. Visual Quantification of Proteins via Conventional ELISA. It is known that proteins are the most common biomarkers for clinical diagnosis,34 and ELISA is one of the most popular techniques for the determination of proteins. Normally, ELISA is conducted in the hospital with sophisticated apparatus such as the automatic microplate readers. However, the poor portability and high cost of microplate reader (U.S. dollars; >4 000) prevent its utility for personal healthcare at home. Herein, we demonstrated that protein biomarkers can be detected without any sophisticated devices by the combination of NEQ assay and the conventional sandwich-format enzyme-linked immunosorbent assay (NEQELISA for short). Because of the good portability and affordability, the proposed NEQ-ELISA could be a good supplement to conventional ELISA for the detection of disease biomarkers out of the hospital. Scheme 1 shows the mechanism of the proposed NEQELISA for visual quantification of arbitrary proteins. The
This reaction can oxidize Au(0) to Au(III) and is used for producing AuNRs of different aspect ratios with narrow size distributions. The corresponding reaction equation is given below:30 2Au + 3H 2O2 + 6H+ + 8Br − → 2[AuBr4]− + 6H 2O (1)
It is worthy to note that when the concentration of H2O2 was relatively low (e.g., less than 100 mM), the oxidation rate was rather slow at room temperature. It spent more than 12 h to fully oxidize the AuNRs into Au(III).31 Therefore, the reaction rate must be accelerated for sensing applications. Herein, Fenton’s reaction was utilized to accelerate the oxidation rate of AuNRs. The corresponding reaction equations are listed below (eqs 2 and 3):32,33 Fe 2 + + H 2O2 → Fe3 + + •OH + OH−
(2)
Fe3 + + H 2O2 → Fe 2 + + •OOH + H+
(3)
2+
In these reactions, Fe acts as a catalyst for disproportionation of H2O2 to create two different oxygen-radical species of • OH and •OOH, respectively. The oxidizability of •OH is much stronger than H2O2. Thus, the oxidation rate of AuNRs could be significantly improved. Our investigation revealed that it spent less than 10 min to completely oxidize AuNRs into [AuBr4]− by using the proposed method even in a low initial concentration (e.g., 5.0 mM) of H2O2 (see Figure S1). Figure 1a shows the H2O2 concentration dependent colorimetric responses of the NEQ assay. In the absence of H2O2, the longitudinal plasmon band of AuNRs located at ∼765 nm (curve 1 of 14). After the addition of H2O2, the longitudinal plasmon band was blue-shifted and the peak intensity was decreased as well. This result indicated that AuNRs were shortened in the presence of H2O2. The amount of peak shift was closely related to the concentration of H2O2. Figure 1b shows representative photographs of corresponding AuNRs solutions after the addition of different amounts of H2O2. It was clearly shown that upon the addition of different concentration of H2O2, the solution color varied from brownish to gray, cyan, green, blue, violet, red, colorless, and yellow. These colors covered nearly the whole visible range from 400 to 760 nm and can be easily distinguished by the naked eye. Some typical TEM images representing the etching process of AuNRs are shown in Figure 1c. The original AuNRs have an average aspect ratio of ∼3.8 and a brownish native color. When the aspect ratio decreased to ∼3.1, the color of the AuNRs solution was changed into cyan. Further decrease in aspect ratio to ∼2.3 produced a blue color solution. The red color solution corresponding to the quasi-spherical Au nanoparticles, and the longitudinal plasmon band disappeared in this case. A stepwise increase of the concentration of H2O2 turned the solution color from red to pink and then colorless (see Figure 1b). The colorless solution indicated that all the Au nanoparticles disappeared, and Au0 was oxidized into colorless [AuBr2]−. AuBr2− was eventually oxidized into yellow [AuBr4]− when further increasing the amount of H2O2. This kind of multicolor variation was easily to be distinguished by the naked eye. Thus, it could be utilized for NEQ detection of H2O2. It can be seen from Figure 1 that a broad dynamic range of 0.05 to 5.0 mM was observed for the detection of H2O2 with the naked eye. It is worthy to note that H2O2 is widely involved in a number of chemical and biological reactions, thus the above phenomenon can be utilized for visual quantification of those
Scheme 1. Principle of the Proposed NEQ-ELISA for Visual Quantification of Proteins
proposed sensing strategy involves a standard sandwich-format immunoassay process. Briefly, the first step of this assay was a typical sandwich-format reaction. A monoclonal antibody immobilized on the surface of the 96-well plates was utilized for the capture of the antigen (target molecule/protein). Then a catalase labeled polyclonal antibody was binding with the antigen to form the antibody−antigen-second antibody sandwich complex on the plate surface. The second step was an enzymatic reaction. Catalases attached on the plate surface acted as catalysts to catalyze the decomposition of H2O2 to water and oxygen. The third step was the signal generation reaction. Fe2+ ion acted as a catalyst for disproportionation of H2O2 to produce a more reactive radical species (•OH). Then • OH was quantitatively reacted with AuNRs to make it shorter. As a result, the final color of the solution was closely related to the concentration of target proteins. To show the feasibility of the proposed NEQ-ELISA for the detection of the disease biomarker, semiquantitative determination of human alpha fetoprotein (AFP) was selected as a target molecule for the sensor demonstration. AFP has been widely accepted as a diagnostic biomarker to detect the early stage of human hepatocellular carcinomas (HCC).35−37 Herein, monoclonal antihuman AFP antibody (produced in mouse) was used as the capture antibody, and polyclonal antihuman AFP antibody was used as the catalase-labeled antibody. Figure 3230
DOI: 10.1021/acs.analchem.5b04621 Anal. Chem. 2016, 88, 3227−3234
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standard solution was designated as the sample concentration; if the solution color was located in between those of two standard solutions, then the AFP concentration of the sample was designated as the average value of the two standard solutions. On the basis of our approach, we have shown the detection results of eight clinical samples in Figure S1 and the results were compared with those obtained from the hospital (see Table S1). Matched-pair t test was used to analyze the consistency of the two approaches, and the results showed that no significance can be claimed (probability = 0.05). Importantly, AFP was quantitatively determined by the naked eye using the proposed NEQ-ELISA, while the results obtained from the hospital were conducted with an expensive chemiluminescent immunoassay (CLIA) system. Visual Quantification of Small Molecules via Competitive ELISA. In many cases, e.g., on-site environmental monitoring and food inspection, the targets could be small molecules rather than proteins. Thus, the visual quantification of these small molecules was highly desired for these applications. However, different from most of the proteins which may bind to different antibodies simultaneously, the small molecule usually can only bind to one antibody. Thus, conventional standard sandwich-format immunoassay is not suitable for the detection of small molecules. Herein, we demonstrated the combination of NEQ assay with a competitive ELISA (NEQ-c-ELISA for short) for visual quantification of small molecules. The detection of a food safety-related substance, aflatoxin B1 (AFB1), was selected as a model for the demonstration. The mechanism of this NEQ-cELISA was shown in Scheme 2. Briefly, the AFB1 in the sample
Figure 2. UV−vis spectra (a) and corresponding photographs (b) of the proposed NEQ-ELISA for visual quantification of AFP. The concentrations of AFP are 0 (control), 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, and 120 ng/mL, respectively, corresponding to the sample numbers 1 to 24.
Scheme 2. Schematic Diagram of the Proposed NEQ-cELISA
containing different amounts of AFP. It should be noted that in the absence of AFP, AuNRs were completely oxidized into [AuBr4]− and the solution color changed from brownish to yellow (sample no. 1 in Figure 2b). This could be helpful to avoid false negative to some extent since the negative sample would also generate a significant color change. In view of the widely accepted cutoff value as 20 ng/mL for the early diagnosis of HCC,38 we intentionally controlled the enzymatic reaction time within 10 min, so that the solution color is transferred from colorless to pink when the concentration of AFP is higher than 20 ng/mL. On the basis of our observation, the color change from colorless to pink is the most sensitive ones for visual inspection. The visual inspection dynamic range for AFP is from 0 to 120 ng/mL, and the visual LOD was 5.0 ng/mL. These analytical figures of merit can fully satisfy the requirements of clinical diagnosis of HCC. Next, we demonstrated the applicability of the proposed NEQ-ELISA for visual inspection of AFP in human serum. Visual quantification of AFP was achieved by the procedures listed: (1) Each sample was tested by the same procedures used for the detection of standard AFP solutions as stated above. (2) The color of each sample solution was compared with those of the standard samples (solutions) shown in Figure 2b. If the color of a sample was close to that of a standard sample (solution), the corresponding AFP concentration of the
and catalase-linked AFB1 in the reaction solution were competitively bound to the anti-AFB1 antibody. We have fixed the concentration of catalase-linked AFB1 in the reaction solution, so that the number of catalase-linked AFB1 immobilized on the microplate was reversely proportional to the concentration of AFB1 in the test sample. Therefore, the amount of H2O2 consumed by the enzymatic reaction is also reversely proportional to the concentration of AFB1. Consequently, after the addition of AuNRs, the amount of LSPR peak shift generated by H2O2-etching was proportional to the concentration of AFB1 in the test sample. It should be noted that this phenomenon was contrary to the method for visual inspection of proteins proposed above. Figure 3a shows the representative photographs for colorimetric responses of the proposed NEQ-c-ELISA to different concentrations of AFB1. The color of solution varied vividly with the increase of the concentration of AFB1. These vivid colors can be easily distinguished at a glance, thus the visual quantification of AFB1 was successfully performed. In 3231
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the proposed visual inspection are 0.065, 0.10, 0.17, 0.23, 0.29, and 0.41 ng/mL, respectively. Good consistency was observed between the concentrations of AFB1 spiked and found, indicating the high feasibility of the proposed NEQ-c-ELISA for real sample assay. Visual Quantification of Nucleic Acids via Magnetic Immunoassay. Magnetic immunoassay (MIA) is another technique that has been widely used for clinical diagnostics, environmental monitoring, and food inspection.39,40 Herein, we extended our demonstration that AuNRs were also an ideal color indicator to conduct naked-eye semiquantitative magnetic immunoassay (NEQ-MIA). Visual quantification of microRNA21 (miRNA-21) with the naked eye was selected as an example to show the feasibility of the proposed approach. The miRNA21 was recognized as a potential tumor marker related to multiple tumors such as brain tumor, colorectal cancer, breast cancer, and pancreatic cancer.41−43 Scheme 3 shows the schematic immunoassay mechanism of the NEQ-MIA. A LNA modified DNA probe was immobilized on the surface of the magnetic bead (MB) to capture the target miRNA, and the other LNA modified DNA labeled with catalase was utilized as an enzyme-label probe to generate the colorimetric signal. LNAs were presented in both probes in order to improve the hybridization efficiency.44 The sequences and modifications of oligonucleotides used in this work was presented in Table S2. Next, we demonstrated the visual determination of the concentration of miRNA-21 by the proposed NEQ-MIA (Figure 4a). Vivid color changes were observed in response to different concentration of miRNA-21 ranging from 5.0 × 10−15 M to 1.0 × 10−11 M, thus visual determination of the concentration of the miRNA-21 was accomplished. Noting that the color of the control sample (sample no. 1) was close to that of the solution added with single-base mismatch strand (10 pM, sample no. 2). These results indicated that the proposed NEQMIA can effectively distinguish the complementary target from those with single-base mismatch strand. Finally, we showed the feasibility of the proposed approach for visual inspection of miRNA-21 in the HeLa cell extractive
Figure 3. NEQ-c-ELISA for visual quantification of AFB1. (a) Photographs of the NEQ-c-ELISA in response to different concentration of AFB1 in standard solutions. The concentrations of AFB1 standard solutions are 0 (control), 0.02, 0.05, 0.08, 0.10, 0.12, 0.14, 0.16, 0.18, 0.20, 0.22, 0.24, 0.26, 0.28, 0.30, 0.32, 0.34, 0.36, 0.38, 0.40, and 0.42 ng/mL, respectively, corresponding to the sample numbers 1 to 21; (b) photographs of the NEQ-c-ELISA for the detection of AFB1 spiked in rice extracts. Concentrations of AFB1 spiked were 0.06, 0.12, 0.18, 0.24, 0.30, and 0.40 ng/mL, respectively, corresponding to the sample numbers 1 to 6.
order to show the applicability of the proposed approach for the detection of AFB1 in real samples, rice samples were spiked with AFB1 and were analyzed (Figure 3b). Visual quantification of the concentrations of AFB1 in these samples was conducted with the same procedures used for the detection of proteins discussed above. The concentration of AFB1 spiked in the rice extraction (10 times dilution) are 0.06, 0.12, 0.18, 0.24, 0.30, and 0.40 ng/mL, respectively, while the concentration found by Scheme 3. Schematic Diagram of the Proposed NEQ-MIA
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Figure 4. Utility of NEQ-MIA for visual quantification of miRNA-21. (a) Photographs of the NEQ-MIA in the presence of different concentrations of miRNA-21. The concentrations of miRNA-21 for sample numbers 1 to 26 are as follows: control (0 M), single-base mismatch (1.0 × 10−11 M), 5.0 × 10−15 M, 1.0 × 10−14 M, 2.0 × 10−14 M, 4.0 × 10−14 M, 6.0 × 10−14 M, 8.0 × 10−14 M, 1.0 × 10−13 M, 1.2 × 10−13 M, 1.5 × 10−13 M, 2.0 × 10−13 M, 3.0 × 10−13 M, 4.0 × 10−13 M, 6.0 × 10−13 M, 8.0 × 10−13 M, 1.0 × 10−12 M, 1.5 × 10−12 M, 2.0 × 10−12 M, 3.0 × 10−12 M, 4.0 × 10−12 M, 5.0 × 10−12 M, 6.0 × 10−12 M, 7.0 × 10−12 M, 8.0 × 10−12 M, and 1.0 × 10−11 M, respectively; (b) photographs of the NEQ-MIA for the detection of HeLa cell lysates. The amount of HeLa cells used for sample number 1 to 4 is 5.0 × 103, 5.0 × 104, 5.0 × 105, and 5.0 × 106, respectively.
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(Figure 4b). Significant color variations were observed when the number of HeLa cells changed from 5.0 × 103 to 5.0 × 106. Meanwhile, distinguishable color variation was observed when detecting miRNA-21 extracted from 5000 HeLa cells. These results strongly indicated the potential applicability of the proposed NEQ-MIA for biological and biomedical applications.
*E-mail:
[email protected]. Phone: +86-591-22866164. Fax: +86-591-22866135. Author Contributions ∥
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X.M. and Z.C. contributed equally to this work.
Notes
CONCLUSIONS In conclusion, this work demonstrated that AuNRs were an ideal chromogenic substrate to conduct multicolor immunoassays. Our investigation reveals that in the presence of Fe2+, H2O2 can quantitatively and efficiently oxidize AuNRs into Au(III). A varied concentration of H2O2 could change the color of AuNRs solution from brownish to gray, cyan, green, blue, violet, red, colorless, and yellow, respectively. These vivid colors can be easily distinguished with the naked eye. The combination of the vivid color display of AuNRs with those immunoassay approaches allows us to develop a number of NEQ assay methods, e.g., NEQ-ELISA, NEQ-c-ELISA, and NEQ-MIA. These NEQ assay methods have been demonstrated for visual quantification of proteins, nucleic acids, and small molecules with the naked eye. To the best of our knowledge, this is the first work that devoted to the development of multiple NEQ assays for sensitive detection of a broad range of analytes. We expect that the proposed NEQ assay could find its applications in those areas that expensive and bulky equipment are not applicable, e.g., on-site environmental monitoring, food inspection, and in-home personal healthcare.
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AUTHOR INFORMATION
Corresponding Author
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support of the National Natural Science Foundation of China (Grants 21277025, 21275031, 21375021, and 21575027), the Program for Changjiang Scholars and Innovative Research Team in University (Grant No. IRT15R11), and the Foundation of Fujian Educational Committee (Grants JA12039 and JA13024).
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b04621. Experimental details, the effect of incubation time on the color changes, reproducibility of the proposed method for visual inspection of AFP, photographs of the sensor for the detection of eight clinical samples, comparison of the proposed method and CLIA for the detection of AFP in eight clinical samples, MiRNA and DNA oligonucleotides used in this work, and comparison of the proposed methods with other colorimetric approaches (PDF) 3233
DOI: 10.1021/acs.analchem.5b04621 Anal. Chem. 2016, 88, 3227−3234
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