A Self-Contained Chemiluminescent Lateral Flow Assay for Point-of

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A Self-Contained Chemiluminescent Lateral Flow Assay for Point-ofCare Testing Jinqi Deng,†,‡,§ Mingzhu Yang,† Jing Wu,† Wei Zhang,*,†,‡ and Xingyu Jiang*,†,‡

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†

Beijing Engineering Research Center for BioNanotechnology and CAS Key Lab for Biological Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, No. 11 Zhongguancun Beiyitiao, Beijing 100190, China ‡ Sino−Danish College, Sino−Danish Center for Education and Research, University of Chinese Academy of Sciences, Beijing 100049, China § Department of Pharmacy, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark S Supporting Information *

ABSTRACT: Immunoassays whose readouts rely on chemiluminescence are increasingly useful for a broad range of analytical applications, but they are rarely made into point-of-care (POC) format because of the complex reagents required (some reagents have to be stored in low temperatures, and some reagents have to be freshly made right before the assay). This study reports a selfcontained chemiluminescent lateral flow assay (CLFA), which prestores all necessary reagents. This CLFA contains three parts: the normal lateral flow assay (LFA) strip, the chemiluminescence substrate pad, and the polycarbonate (PC) holder. On the LFA strip, we simultaneously labeled horseradish peroxidase (HRP) and antibody on the gold nanoparticles (AuNPs) for the conjugate pad. For the substrate pad, we used sodium perborate as the oxidant and lyophilized the chemiluminescence substrate on the glass fiber, which allows long-term storage. After the transfer of substrate from the substrate pad to the nitrocellulose (NC) membrane, we captured the chemiluminescence signal for the quantification of the targets. The HRP on the AuNPs can amplify the chemiluminescence signal efficiently. We used this CLFA system to detect both macromolecules and small molecules successfully. This self-contained and easily processable device is exceedingly appropriate for rapid detection and is a convenient platform for POC testing.

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conventional GLFA out of many areas. Many efforts have been put to improve the performance of LFA.13−15 For example, magnetic nanoparticles (MNPs)16 and DNAzyme17 were employed to amplify the signals due to their excellent catalytic activity. Quantum dots (QDs)18,19 and upconversion nanoparticles (UCNPs)20,21 were applied for quantitative detection of target molecules. However, most of these methods need multiple operations, which increase the complexity of the whole detection process. To overcome the obstacles without abandoning its simplicity, some researchers tried to reconstruct the structure of the LFA and integrate different functional components,22 but this alternative needs expensive equipment and complex fabrication steps and increases the difficulty for fabrication, which are additional obstacles for POC platforms. Chemiluminescence (CL) is broadly adopted in the fields of detection and diagnosis.23−26 Compared with other optical methods, the CL method has high sensitivity and high signalto-noise ratio. The instruments for readout of the CL signal are simpler than other optical instruments. The H2O2/luminol CL system is widely used in enzyme-linked immunosorbent assays

oint-of-care (POC) testing offers powerful tools for disease diagnosis,1−5 environmental analysis,6,7 and food safety monitoring.8,9 Paper-based devices,10,11 especially lateral flow assays (LFA),12 are widely used POC platforms. Conventional gold nanoparticles (AuNPs)-based LFA (GLFA) consists of five parts: the sample pad, the conjugate pad, the nitrocellulose (NC) membrane, the absorbent pad, and the poly(vinyl chloride) (PVC) sheet. The conjugate pad contains AuNPs that conjugate with antibodies which can recognize targets in the sample. The NC membrane is dispensed with antibodies that can recognize either targets (for the test line) or antibodies on the AuNPs (for the control line). The sample introduced on the sample pad flows to the absorbent pad because of the capillary effect. When the sample flows through the conjugate pad, AuNPs move along with the sample and antibodies on the AuNPs recognize the targets. The AuNPs are immobilized on the NC membrane because of the immunoreaction between the antibodies and antigens. The existence of targets causes both the test line and the control line to turn red, while only the control line becomes red if the targets are absent. GLFA is quite suitable for POC testing due to its great intrinsic qualities including robustness, specificity, simple operation, and fast response. However, low sensitivity and lack of the quantification ability have already kept © XXXX American Chemical Society

Received: April 5, 2018 Accepted: July 2, 2018

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DOI: 10.1021/acs.analchem.8b01543 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry (ELISA)27−29 and LFA30,31 as the H2O2/luminol system can be catalyzed by horseradish peroxidase (HRP), which is one of the most frequently used enzymes labeled on the antibody. For the H2O2/luminol system, the two liquid components need to be stored individually and mixed together when used. The storage and mixing of the liquid components increase the operations of detection. This complication prevents CL from becoming useful for POC testing. So setting up a selfcontained system will dramatically broaden the application of CL. Lyophilization is a good choice for self-contained POC platforms,32,33 but it is unsuitable for the H2O2/luminol system as H2O2 will decompose during lyophilization. In this study, we developed a self-contained and easily processable chemiluminescent lateral flow assay (CLFA) for POC testing. This CLFA contains three parts: the LFA strip, the substrate pad, and the polycarbonate (PC) holder (Scheme 1). The LFA strip was similar to that of conventional GLFA, except that the AuNPs on the conjugate pad were labeled with antibody and HRP simultaneously. On the substrate pad, we lyophilized the mixture of the CL substrate on the glass fiber. The oxidant we chose was not H2O2 but one of its analogues, sodium perborate, which can also react with luminol to produce a strong CL signal. After the general steps of GLFA and qualitative analysis by the naked eye, we dissolved the substrate with deionized water and covered the substrate pad on the LFA for a short time to transfer the substrate to the NC membrane. The substrate mixture reacted under the catalysis of HRP and generated a CL signal for quantitative detection. We applied this CLFA to detect both marcomolecules [using α-fetoprotein (AFP) as a model] and small molecules [using folic acid (FA) as a model]. Due to the amplification of HRP, we improved the sensitivity of marcomolecules and broadened the detection range of small molecules.

Scheme 1. Scheme of the CLFAs for the Detection of Targetsa

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EXPERIMENTAL SECTION Materials and Equipment. Horseradish peroxidase, bovine serum albumin (BSA), and chloroauric acid (HAuCl4· 3H2O) were from Sigma-Aldrich. AFP, anti-AFP monoclonal capture antibody, anti-AFP monoclonal detection antibody, and goat antimouse secondary antibody were from Beijing Eastmo Biotechnology Co., Ltd. FA, FA-conjugated BSA (FA− BSA), and anti-FA monoclonal antibody were from Beijing Zeyang Biotechnology Co., Ltd. Potassium persulfate, sodium persulfate, sodium percarbonate, sodium perborate, hydrogen peroxide, luminol, and 4-indophenol (PIP) were from Shanghai Macklin Biochemical Co., Ltd. The NC membrane was from Millipore. The PVC sheet, the conjugate pad, the sample pad, and the absorbent pad were from Shanghai Kinbio Technology Co., Ltd. Phosphate-buffered saline (PBS, pH 7.4, 0.01 M) was from Solarbio. Other reagents were of analytical grade. All chemicals were used as received without further purification. We employed an XYZ dispense platform (XYZ3060, Biodot, U.S.A.) and CM4000 slitter (Biodot, U.S.A.) for the preparation of the CLFA. We employed a scanning electron microscope (SEM, SU-8220, Hitachi, Japan) and energydispersive spectrometer (EDS, EV370X, Horiba, Japan) for the characterization of the substrate pad. We employed a multimode plate reader (Enspire, PerkinElmer, U.S.A.) and a CL analyzer customized by the National Center for NanoScience and Technology (Beijing, China) for the collection of the CL signals.

a

Sample is introduced on the sample pad and moves to the absorbent pad. When sample flows through the conjugate pad, the targets are recognized by the detection antibodies on the AuNPs. With sample flowing through the NC membrane, targets and detection antibodies, as well as AuNPs, are immobilized on the NC membrane. After the appearance of the red lines because of the aggregation of AuNPs, the substrate pad containing lyophilized substrate is covered on the LFA strip for the transfer of the CL substrate. Then the substrate pad is removed, and the CL signal is captured for quantitative detection of targets.

Comparison of the CL Activity of Different Peroxides. PIP and luminol were dissolved in NaOH solution and were diluted in PBS. Potassium persulfate, sodium persulfate, sodium percarbonate, and sodium perborate were dissolved in deionized water. Hydrogen peroxide was diluted in deionized water. HRP was dissolved in PBS. We added 50 μL of 10 mM luminol solution, 50 μL of 10 mM PIP solution, and 50 μL of HRP solution with different concentrations (0, 10, 20, 50, 100, 200, 500, 1000 ng/mL) in the 96-well plate. After the addition of different peroxides (100 μL, 5 mM), we measured the CL intensity immediately. To compare the CL activity of “solidified” substrate, we mixed 1 mL of 100 mM luminol, 1 mL of 100 mM PIP, and 2 mL of 50 mM peroxides together and saturated the glass fiber with the mixture. We lyophilized the glass fiber and cut it into squares (5 mm × 5 mm). We added 200 μL of deionized water and 50 μL of HRP solution with different concentrations (0, B

DOI: 10.1021/acs.analchem.8b01543 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

For the sensitivity evaluation of the FA detection, the concentrations of FA were 50, 20, 10, 5, 2, 1, 0.5, 0.2, 0.1, and 0 ng/mL. The procedure was the same as above. For the milk powder sample detection, we dissolved and diluted the milk powder samples with PBS. The other procedure was the same as above. The milk powder samples were gotten from Heilongjiang Wondersun Dairy Co., Ltd.

10, 20, 50, 100, 200, 500, 1000 ng/mL) in the 96-well plate. We put the squares in the wells and measured the CL intensity. The substrate was stored at a dark, dry, and sealed condition at 4 °C for the stability test. The procedure was the same as above. Preparation of the AuNPs−Protein Conjugates. AuNPs were prepared according to the literature. Briefly, 100 mL of HAuCl4 solution (0.01%, mass fraction) was heated under reflux with stirring, followed by the addition of 1.2 mL of trisodium citrate solution (1%, mass fraction). The mixture was kept heating for another 15 min. The color change of the mixture proved that AuNPs were synthesized successfully. After cooling, the solution containing AuNPs was filtered and stored at 4 °C for further use. We adjusted the pH of the solution containing AuNPs to 8.5 using 25 mM K2CO3 solution. We added antibody (50 μg/ mL) and HRP (250 μg/mL) to the solution, and the mixture was incubated for 30 min. We used BSA solution (final concentration 1%) to block the AuNPs for 30 min. The mixture was centrifuged twice (9500 rpm, 25 min). Finally, we suspended the AuNPs−protein conjugates in the recovery solution and stored the conjugates at 4 °C for further use. Fabrication of the CLFAs. The CLFAs consist of seven components: the sample pad, the conjugate pad, the substrate pad, the NC membrane, the absorbent pad, the PVC sheet, and the PC holder. We saturated the conjugate pad with the AuNPs−protein conjugate solution and freeze-dried it. We saturated the substrate pad with CL substrate and freeze-dried it. We used a dispenser to dispense the capture antibody (1.7 mg/mL) and the goat antimouse secondary antibody (2 mg/ mL) on the NC membrane and dried the NC membrane at 37 °C for 1 h. We assembled the sample pad, the conjugate pad, the NC membrane, and the absorbent pad on a PVC sheet and pasted the substrate pad on another PVC sheet. The PVC sheets were cut into strips with a width of 4 mm. The strips were kept under a dark, dry, and sealed condition at 4 °C. Procedure of the CLFAs. To evaluate the sensitivity of the CLFAs for AFP detection, we added 100 μL of AFP solution with different concentrations (200, 100, 50, 20, 10, 5, 2, 1, 0.5, and 0 ng/mL) to the sample pad. After 15 min, we added 20 μL of deionized water to the substrate pad and covered the substrate pad on the NC membrane for 10 s. We used a customized CL analyzer to capture the CL signals. The exposure time was 60 s. The images were analyzed using ImageJ software. All the AFP sample solutions were tested three times independently. To test the selectivity of the CLFA method for AFP detection, we used the CLFA to detect six different proteins: AFP, carcinoembryonic protein (CEA), C-reactive protein (CRP), procalcitonin (PCT), interleukin-6 (IL-6), and immunoglobulin G (IgG). All the concentrations of the six proteins are 100 ng/mL. The procedure was the same as above. For AFP serum detection, we diluted the AFP serum samples by 2 times using PBS with 30% fetal bovine serum (FBS). We added 100 μL of diluted serum sample to the sample pad. The other procedure was the same as above. The serum samples were collected from the Chinese PLA General Hospital (Beijing, China) in accordance with the hospital guidelines (The Ethics Guidelines for Research Involving Human Subjects or Human Tissue from the Chinese PLA General Hospital).

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RESULTS AND DISCUSSION Comparison of the CL Activity of Different Peroxides. We tested the reaction activity of luminol with several different peroxides. The principle of the H2O2/luminol CL system is that O2 produced by H2O2 oxidizes luminol to form an unstable intermediate, and this intermediate converts into 3aminophthalic acid with a photon (λ = 425 nm) released (Figure S1).34,35 So we tried other solid peroxides that can produce H2O2 upon contact with water. We chose four different peroxides: potassium persulfate, sodium persulfate, sodium percarbonate, and sodium perborate. All the four peroxides can react with luminol and produce CL signals (Figure 1A). Compared with H2O2, potassium persulfate and sodium persulfate produce weak CL signals. Sodium percarbonate and sodium perborate can produce stronger CL signals than H2O2. We think that different peroxides have different reactivities with luminol. We adopted sodium percarbonate and sodium perborate for the further experiments.

Figure 1. Comparison of the CL activity of different peroxides. (A) Comparison of the CL activity of five peroxides in solution. (B) Comparison of the CL activity of hydrogen peroxide, sodium perborate, and sodium percarbonate after lyophilization and redissolution. (C) The CL activity of the substrate pad with different storage times under 4 °C. The error bars are from three independent repeats.

Inspired by the conjugate pad used in LFA, we used a glass fiber and lyophilization method to solidify the substrate mixture. We used SEM to characterize the glass fiber (Figure 2). The morphology change of the glass fiber proved that substrate mixture was freeze-dried on the glass fiber successfully. We also used EDS to characterize the glass fiber that contained sodium perborate (Figure S2). The signals of B (from sodium perborate), P (from PBS), and I (from PIP) proved the existence of the CL substrate on the glass fiber. C

DOI: 10.1021/acs.analchem.8b01543 Anal. Chem. XXXX, XXX, XXX−XXX

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and captured the CL signals of the strips for quantitative analysis. Similar to the colorimetric results, the CL intensity of the test line decreases when the AFP concentrations decrease from 200 to 0.5 ng/mL (Figure 3B). A linear relationship is established with the concentration from 1 to 200 ng/mL, and the equation is Y = 11733.02X + 2123.98 (X = Lg(CAFP), R2 = 0.97) (Figure 3C). The limit of detection (LOD) for AFP detection is calculated to be 0.27 ng/mL (LOD = 3σ/M, σ = 180.17, M = 1964.90). The LOD of the CL-based readout is 20 times lower than that of the naked-eye readout. This is due to the amplification of HRP labeled on the AuNPs.

Figure 2. SEM images of glass fiber before (A) and after (B) lyophilization with CL substrate.

Then, we tested the CL activity of the freeze-dried substrate. After lyophilization, the CL intensity of the substrate that contained H2O2 or sodium percarbonate decreased significantly, but the substrate that contained sodium perborate could produce strong CL signals (Figure 1B). We think it is because of the structural difference of these three peroxides. H2O2 is an unstable liquid. During the lyophilization, H2O2 will decompose into H2O and O2. So the CL signal of H2O2/ luminol essentially became weak after lyophilization. Sodium percarbonate (2NaCO3·3H2O2) is a complex of Na2CO3 and H2O2. When dissolved in water, it will convert into Na2CO3 and H2O2. The performance of sodium percarbonate during lyophilization was similar to that of H2O2, and the CL intensity after lyophilization decreased, too. For sodium perborate (Na2B2(O2)2(OH)4), the peroxy group is bonded to two boron atoms. This structural difference slows down the decomposition rate of sodium perborate, and the CL intensity of sodium perborate after being freeze-dried is much higher than that of H2O2 and sodium percarbonate. We also stored the sodium perborate/luminol substrate mixture under 4 °C to test its stability (Figure 1C). The CL signal did not show significant change even after 15 days. This result demonstrated that the substrate mixture had great stability under 4 °C. We used sodium perborate as the oxidant of the CL system for the further experiments. Detection of AFP with CLFAs. We employed the CLFAs to detect AFP, a cancer biomarker. The detection of AFP is based on the sandwich ELISA principle. A pair of antibodies, called capture antibody and detection antibody, is used for the sandwich ELISA. Capture antibody is immobilized on the substrate for the capture of AFP. Detection antibody, usually labeled with enzymes, can recognize AFP and generate signals for detection. We dispensed anti-AFP capture antibody and secondary antibody on the NC membrane. We labeled antiAFP detection antibody and HRP together on the AuNPs with mass ratio 1:5.14 The CL signal was captured with a customized CL analyzer (Figure S3). This CL analyzer is 17.5 cm × 35 cm × 18.5 cm, and it costs around $5000. We investigated the duration of the CL signals (Figure S4). The CL signals began to attenuate after 3 min, and could not be captured after 10 min. In the following experiments, the CL signals were captured within 3 min after the removal of the substrate pad. We supplied a series of AFP solutions to the strips to test the sensitivity of the CLFAs. The color of the AuNPs at the test line decreased as concentrations of the AFP changed from 200 to 5 ng/mL (Figure 3A). The red line could not be distinguished by the naked eye when the AFP concentration was lower than 5 ng/mL. We dissolved the substrate with deionized water, covered the substrate pads on strips for 10 s,

Figure 3. Results of AFP detection with CLFAs. (A) The naked-eye readout and CL signals of AFP detection. (B) The relationship between the CL intensity and the AFP concentrations. (C) The linear range for AFP detection using CLFAs.

We employed six different proteins (AFP, CEA, CRP, PCT, IL-6, and IgG) to investigate the selectivity of CLFAs. Among the six proteins, AFP shows positive results for both naked-eye readout and CL readout, while all of the other five proteins show negative results (Figure 4A), which proves that the CLFAs have high selectivity. This high selectivity is due to the high affinity between antibody and antigen.

Figure 4. Performance evaluation of CLFA for AFP detection. (A) Selectivity test of CLFA using six proteins. (B) Comparison of the CLFA method and ECL method for AFP detection.

To test the performance of the CLFAs in clinical use, we used the CLFAs to detect AFP in 27 real serum samples. The AFP levels of the serum samples were determined by an automated electrochemiluminescence (ECL) immunoassay analyzer (Roche ECL, Cobas E411, Roche Diagnostics). The clinical cutoff value for AFP detection is 5 ng/mL. Samples 1− 9 show negative results with the two methods, and samples 10−27 are both positive (Table S1). The further comparison of the results between CLFA and ECL shows a great linear D

DOI: 10.1021/acs.analchem.8b01543 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry Table 1. Comparison of the Analytical Performance of CLFA with Other Methods for AFP Detection method

LOD (ng/mL)

time (min)

readout

self-contained

fabrication

ref

CLFA GLFA QD-ICTSa electrochemical LFICb wax-printed LFIAc

0.27 20 3 1.0 0.1

18 15 15 20 10

quantitative semiquantitative quantitative quantitative quantitative

yes yes yes no yes

easy easy easy easy complex

this work 36 37 38 22

a QD-ICTS: QDs-based immunochromatographic test strip (ICTS). bElectrochemical LFIC: electrochemical lateral flow immunochromatographic (LFIC) test strip. cWax-printed LFIA: wax-printed lateral flow immunoassay.

relationship (R2 = 0.98) (Figure 4B), which demonstrates that the CLFAs have the similar quantitative capability as the ECL method. We compared several reported lateral flow platforms for AFP detection (Table 1). Conventional GLFA can only be used for qualitative and semiquantitative analysis, and its sensitivity is unsatisfactory. Many efforts have been put to improve the analytical performance of lateral flow assays. Compared with other lateral flow platforms, our CLFA can reach high sensitivity due to the high signal-to-noise ratio of CL and the amplification of HRP. Recently, a wax-printed LFA is reported with quite high sensitivity,22 but the requirement of the wax-printed machine limits its application. In contrast, our CLFAs have excellent analytical performance and they are easy to fabricate and employ. Detection of FA with CLFAs. To show that this approach is also applicable to other variants of LFA, we utilized CLFAs to detect FA. FA is a small molecule that takes roles in many important biochemical reactions, including the synthesis of purine and pyrimidine and the metabolism of amino acids. Besides, FA is an indispensable substance for the growth and development of the fetus. Many milk powders contain FA as one of the nutrients. Unlike AFP, the detection of FA is based on the competitive ELISA principle. We conjugated the anti-FA antibody on the AuNPs and dispensed FA−BSA on NC membrane for the test line. We supplied a series of FA standard solutions to the sample pad and waited for 15 min for the immunoreaction. With the increase of the FA concentration, the color of the test line decreased, and the LOD by the naked eye was 0.1 ng/mL. We could not recognize the red line if the concentration was higher than 2 ng/mL (Figure 5A). We also used the CL signal to quantify the FA concentration. The trend of the CL intensity was consistent with that of the colorimetric results, but the signals could still be captured even when the concentration was 50 ng/ mL (Figure 5B). We established a linear relationship between Lg(CL intensity) and Ln[CAFP/(100 − CAFP)] (Y = −0.32X + 2.57, R2 = 0.95) (Figure 5C), in which the concentrations of FA were from 0.5 to 50 ng/mL, and the calculated LOD was 0.22 ng/mL. With CL signals, we broaden the detection range of FA from 0.1−2 to 0.5−50 ng/mL. We further used the CLFAs to detect FA in three milk powder samples. The milk powder samples were dissolved in PBS before detection. The reference values of FA concentrations were determined by the ELISA method. Compared with ELISA results, the recovery rate of the CLFA results is between 95% and 110%, and the coefficient of variation (CV) was less than 15% (Table 2). The agreement between ELISA results and CLFAs results demonstrates this method has great potential in practical applications.

Figure 5. CLFAs for detection of FA by both naked-eye and CL signals. (A) The colorimetric and CL results for FA detection. (B) The corresponding relationship between CL intensity and the concentration of FA. (C) The linear range of detection of FA using CLFAs.

Table 2. Determination of FA in Milk Powder by ELISA and CLFAs sample no.

ELISA (μg/g)

CLFA (μg/g)

recovery rate (%)

CV (%)

1 2 3

176 ± 27.7 89 ± 11.3 80 ± 9.2

170 ± 23.1 95 ± 9.9 84 ± 11.8

96.6 106.7 105.0

13.6 10.4 14.0

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CONCLUSION

We have developed a self-contained lateral flow assay whose readout is chemiluminescence, without having to carry out the complex steps of operations typically required for these assays (such as storing and in situ mixing of luminol with H2O2). This POC platform meets the requirements in analytical performance (such as sensitivity and detection range) of both macromolecules (such as AFP) and small molecules (such as FA). In conjunction with a low-cost CL analyzer, this CLFA would be an easy-to-use and highly sensitive POC device. The key of the success of our method is the use of sodium perborate to release H2O2 controllably. As H2O2 is involved in many important biochemical processes, our strategy can be further applied into different areas such as analysis and chemical/biochemical processes wherever controlled H2O2 release is necessary. E

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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.8b01543.

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Additional experimental data, including the mechanism of the H2O2/luminol CL reaction, photograph of the CL analyzer, duration of the CL signal of the CLFA, EDS images of the glass fiber before and after lyophilization, and the results of detection of AFP in serum samples by ECL and CLFA (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: 86 10 82545620. *E-mail: [email protected]. Phone: 8610 8254 5558. Fax: 86 10 82545631. ORCID

Xingyu Jiang: 0000-0002-5008-4703 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Ministry of Science and Technology of China (2013YQ190467), the Chinese Academy of Sciences (XDA09030305), and the National Science Foundation of China (81361140345, 21535001, 81730051) for financial support.

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DOI: 10.1021/acs.analchem.8b01543 Anal. Chem. XXXX, XXX, XXX−XXX