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Sensitive and Quantitative Detection of C-reaction Protein Based on Immunofluorescent Nanospheres Coupled with Lateral Flow Test Strip Jiao Hu, Zhi-Ling Zhang, Cong-Ying Wen, Man Tang, Ling-Ling Wu, Cui Liu, Lian Zhu, and Dai-Wen Pang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01427 • Publication Date (Web): 02 Jun 2016 Downloaded from http://pubs.acs.org on June 3, 2016
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Analytical Chemistry
Sensitive and Quantitative Detection of C-reaction Protein Based on Immunofluorescent Nanospheres Coupled with Lateral Flow Test Strip Jiao Hu, Zhi-Ling Zhang, Cong-Ying Wen, Man Tang, Ling-Ling Wu, Cui Liu, Lian Zhu, Dai-Wen Pang* Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, The Institute for Advanced Studies, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan 430072, People's Republic of China Correspondence: Professor Dai-Wen Pang College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, P. R. China. Fax: +86-27-68754067 E-mail:
[email protected] ABSTRACT: Sensitive and quantitative detection of protein biomarkers with a point-of-care (POC) assay is significant for early diagnosis, treatment, and prognosis of diseases. In this paper, a quantitative lateral flow assay with high sensitivity for protein biomarkers was established by utilizing fluorescent nanospheres (FNs) as reporters. Each fluorescent nanosphere (FN) contains 332 8 CdSe/ZnS quantum dots (QDs), leading to its superstrong luminescence, 380 folds higher than that of one QD. Then a detection limit of 27.8 pM C-reaction protein (CRP) could be achieved with an immunofluorescent nanosphere (IFN)-based lateral flow test strip. The assay was 257 folds more sensitive than that with a conventional Au-based lateral flow test strip for CRP detection. Besides, the fluorescence intensity of FNs and bioactivity of IFNs were stable during 6 months of storage. Hence, the assay owns good reproducibility (intra-assay variability of 5.3% and inter-assay variability of 6.6%). Furthermore, other cancer biomarkers (PSA, CEA, AFP) showed negative results by this method, validating the excellent specificity of the method. Then the assay was successfully applied to quantitative detection of CRP in peripheral blood plasma samples from lung cancer and breast cancer patients, and healthy people, facilitating the diagnosis of lung cancer. It holds a good prospect of point-of-care protein biomarker detection.
Introduction Rapid, sensitive and quantitative detection of protein biomarkers is becoming crucial for clinical application. Accurate quantification of tumor markers can assist early diagnosis of cancers and determine the extent of diseases. 1-3 Moreover, quantitative detection of protein biomarkers can also aid monitoring of drug therapeutic response in many diseases. For instance, C-reaction protein (CRP) was a marker of acute-phase inflammatory response, and the elevated concentration of inflammation protein CRP will decrease after therapy.4-6 In recent years, different methods for protein detection have been reported, 7-11 and they were typically laboratory tests which suffered from drawbacks of requiring highly trained personnel, expensive instrument or rigid operational protocols. These drawbacks may restrict their application, e.g. for the household use. What’s more, all these methods are time-consuming, which is quite unfavorable for clinical application. Therefore, it is imperative to develop a rapid point-of-care (POC) assay sensitive enough and quantitative for protein biomarker detection. Evidently, among the POC strategies, 12,13 the most well-known method is lateral flow test strip assay, whose unique advantage lies in speediness. Besides, it is low-cost, simple, and 1
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convenient.14,15 More importantly, the strip platform reduces demand for highly trained personnel and expensive equipment. Due to
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these advantages, it has been successfully utilized for detecting not only small molecules,16 nucleic acids,17 but also proteins,18,19 even bacteria.20, 21 To date, the conventional lateral flow test strips are mainly based on gold nanoparticle,19,22,23 which act as a reporter for either qualitative or semiquantitative colorimetric detection. The signals rely on the localized surface plasmon resonance effect of the gold nanoparticles, whose sensitivity is not so high. The above limitations may impede the further application of lateral flow test strips in the fields that need high sensitive and quantitative assay. To solve these problems, fluorescent materials, such as organic fluorophores,24,25 up-conversion phosphors,26,27 and fluorescent quantum dots (QDs),18,28,29 as reporters for lateral flow test strips may be the relatively promising for sensitive and quantitative detection of protein biomarkers since they have inherent signal-to-noise ratios higher than gold nanoparticles used in colorimetry-based detection. Still, these reporters may suffer from photobleaching (organic fluorophores), low emission intensity (up-conversion phosphors) and colloidal instability in complex matrix (up-conversion phosphors, QDs), which lower the sensitivity and stability of the assay. Thereby, new fluorescent reporters that can overcome these limitations are highly needed for lateral flow test strip platform applications in POC quantitative detection of protein biomarkers. The fluorescent nanospheres (FNs) integrated with QDs can meet the requirements for a reporter. Compared to QDs, they are unique, with strong luminescence, convenient manipulation, high stability in complex matrix, and good biocompatibility. 30 In our previous work, based on QDs and nano-γ-Fe2O3, we constructed fluorescent-magnetic nanospheres by embedding them into poly(styrene/acrylamide) copolymer nanospheres. They were successfully used to detect DNA, proteins, bacteria, and cancer cells.31-36 Accordingly, the fluorescent nanosphere (FN) may be an excellent reporter for the lateral flow test strip platform, which enable rapid, sensitive and POC quantitative detection of proteins in complex matrixes. In the present work, a rapid and sensitive assay for quantitative detection of protein biomarker was developed, based on the benefits inherited from both FNs and lateral flow test strip. CRP, a key blood plasma protein, which has robust association with inflammation, tissue damage, and infection, also a non-specific marker of several types of cancer,5,37-39 was used as a model protein. As expected, experimental results indeed demonstrated that the assay can be used to rapidly and quantitatively detect CRP (20 min) with a linear relationship between 0.178 nM and 11.4 nM with a detection limit of 27.8 pM. For CRP detection, the assay was robust with high specificity, strong anti-interference ability and good reproducibility. Thirty-four peripheral blood plasma samples from lung and breast cancer patients and healthy people have been successfully detected by this method, indicating that the immunofluorescent nanosphere (IFN)-based test strip is promising in practice, e.g. simultaneous detection of multiple protein biomarkers or viruses. Experimental section Reagents and Instruments. CRP was from Prospecbio. Mouse monoclonal antibodies against CRP (clone1 and clone2) named MC01, MC02, and goat anti-mouse IgG antibody were purchased from QiTai Technology Co., Ltd (Hangzhou). Human alpha fetoprotein (AFP) was ordered from Linc-Bio Science Co., Ltd. (Shanghai). Carcinoembryonic antigen (CEA) was bought from Fitzgerald Industries International, Inc. Albumin from human serum (HSA), prostate specific antigen (PSA), bovine serum albumin (BSA), FITC-labeled goat anti-mouse IgG (FITC-IgG), and N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) were purchased from Sigma-Aldrich. Fetal bovine serum (FBS) was obtained from Gibco. N-hydroxysuccinimide (NHS) was bought from Thermo. Human peripheral blood plasma samples were supplied by Hospital of Wuhan University and Hubei Cancer Hospital. Ultrapure water (18.2 MΩ·cm) was obtained with a Millipore Milli-Q system and used for the preparation of all solutions. Hydrophobic CdSe/ZnS QDs were purchased from JiaYuan Quantum Dots Co., Ltd (Wuhan). Nitrocellulose membrane, sample pad, absorbent pad and plastic adhesive card were supplied by JieYi BioTech Co., Ltd (Shanghai), and used as provided. The transmission electron microscopy (TEM) images were acquired on a FEI Tecnai G2 20 TWIN electron microscope. Fluorescence emission spectra were collected on a Fluorolog-3 fluorescence spectrometer (HORIBA JOBIN YVON). Dynamic 2
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Analytical Chemistry
light scattering (DLS) measurements were performed on a Zetasizer Nano ZS instrument (Malvern). The concentration of cadmium
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(Cd) atom in the FNs was measured with an Intrepid XSP Radial ICP-OES instrument (Thermo) with a concentric nebulizer and a cinnabar model spray chamber. Fluorescence images were obtained with an inverted fluorescence microscope (Nikon TIU) which equipped with a CCD camera (Nikon DS-Ri1). Fluorescence pictures of lateral flow test strips were recorded with an Alpha Image HP (Alpha). Fabrication of FNs-antibody conjugates. FNs-antibody conjugates (IFNs) were prepared according to the method previously reported by our group.34,35 First, carboxyl-terminated FNs were prepared by embedding hydrophobic CdSe/ZnS QDs into poly(styrene/acrylamide) copolymer nanospheres (Pst-AAm-COOH). Then, FNs were conjugated with MC02 antibody. In brief, approximate 2 mg of FNs were activated firstly in 50 mM EDC and 50 mM NHS in 400 μL of phosphate-buffered saline (PBS) buffer solution (0.01 M, pH=6.8) at room temperature with continuous shaking for 20 min. Afterwards, FNs were separated by centrifugation, and washed with PBS (0.01 M, pH=7.2) twice. Then, they were dispersed in 400 μL of PBS (0.01 M, pH=7.2) to react with MC02 for about 4 h at room temperature with gentle agitation to get the IFNs. After that, the IFNs were blocked with 400 μL of BSA-PBS (0.01M, pH=7.2) at room temperature with gentle agitation. Then IFNs were washed with PBS (0.01 M, pH=7.2) five times. At last, IFNs were dispersed in 100 μL of PBS (0.01 M, pH=7.2) and stored at 4°C until use. Detection of CRP using IFN-based lateral flow test strip assay. The sample (1 μL CRP standard or serum sample) and a certain amount of IFNs were mixed with 1% BSA-PBS (0.01 M, pH=7.4) in a well of 96-well plate (the final volume was 80 μL). Then the mixture was loaded onto the sample pad of the anti-CRP lateral flow test strip, allowing all liquid to be absorbed and migrate along the strip. A few minutes later, images of test zone on the lateral flow test strip were acquired with an EMCCD (Andor iXon DV885K) single photon detector mounted on an inverted fluorescence microscope (Olympus IX 70). Quantitative analysis was performed by scanning the images and measuring the fluorescence intensity immediately with the Andor SOLIS for imaging software. Results and discussion Characterization of the IFNs. According to the strategy developed by our group, illustrated in Figure 1A, FNs were obtained by embedding QDs into Pst-AAm-COOH. The TEM images (Figure 1B insert images) showed that, compared with the Pst-AAm-COOH (insert image a), there were numerous nano-particles widely distributed inside the FN (insert image b), demonstrated the successful fabrication of FNs. From Figure 1B, it can be seen that the size of FNs was uniform. Moreover, Figure 1C showed that the mean diameter of FNs was 272 ± 9 nm (for 200 FNs), and the size coefficient of variation (CV) was calculated to be 3.3%, which proved that the FNs were highly uniform in particle size distribution. Because the embedding method was just a physical process, the organic ligands on the QDs surface were hardly damaged, allowing the QDs to maintain their optical properties to the greatest extent. Hence, the FNs owned superior luminescence. It could be seen that the FNs showed strong fluorescence from the fluorescence microscopic image (Figure 1D). Furthermore, the fluorescence spectra of the QDs and the FNs (Figure 1 E) also indicated that the fluorescence properties of the QDs were still retained in the Pst-AAm-COOH nanospheres.36,40 Similarly, the embedding process also had negligible effect on copolymer nanospheres. DLS (Figure 1G) characterization showed the hydrodynamic size of the FNs in solution was 286.6 nm, a minor increased comparing with the Pst-AAm-COOH (275.1 nm) (Figure 1F). At the same time, the dispersity of FNs was demonstrated by the polydispersity index (0.013), consistent with the monodispersed FNs showed in Figures 1B and 1C, further demonstrating that FNs were well dispersed with perfectly uniform size. Then we calculated that averages of 332 ± 8 QDs (Table S1, Supporting Information) were embedded into one nanosphere (details in Supporting Information S1). Hence, we investigated the fluorescence intensity of FNs compared with hydrophobic QDs at a particle concentration of 5.76 pM (Figure 2A). As shown in Figure 2A, FNs exhibited superstrong luminescence relative to QDs. A linear relationship of fluorescence intensity versus FNs concentration between 1.55 pM and 7.75 pM or fluorescence intensity against hydrophobic QDs concentration between 3.66 nM and 18.3 nM was shown in Figure 2B. The slope of linear 3
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relationship of fluorescence intensity versus FNs concentration was 2.01×106. In contrast, the slope of linear relationship of
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fluorescence intensity versus hydrophobic QDs concentration was 5.28×103. Hence, compared with hydrophobic QDs, the slope for FNs was 380 folds bigger than that for QDs, indicating that one FN can be 380 times brighter than one QD, 41 confirming the stronger luminescence of FNs relative to QDs. With all these predominant characteristics, the FNs were modified with MC02 antibody. To verify the antibody conjugation and calculate the number of MC02 antibody conjugated to one FN, FITC-IgG was employed to react with IFNs by the interaction between the primary antibody and the second antibody, as illustrated in Figure S1A (Supporting Information). As shown in Figure S1B (Supporting Information), the green fluorescence of FITC exhibited on IFNs. After the photobleaching of FITC, only the yellow fluorescence from the IFNs was visible (Figure S1C, Supporting Information). In contrast, no green fluorescence was observed in the control group with FNs (Figure S1D, Supporting Information).32,42 These results demonstrated that IFNs were successfully functionalized with the MC02 antibody. At the same time, a rough number of MC02 antibodies conjugated to each FN was estimated to be about 120 by the fluorescence intensity of FITC-labeled IgG conjugated to the IFNs (Figure S2, Supporting Information). Fabrication of IFN-based lateral flow test strip
As shown in Figure 3A, the test strip consisted of four parts, namely sample pad, nitrocellulose membrane (NC membrane), absorption pad, and plastic adhesive card. The assay employed FNs as reporters to replace Au nanoparticles because of their highly bright fluorescence signal and higher signal-to-noise ratio. Figure 3B illustrated the schematic of CRP detection, which was based on the formation of the IFNs/CRP/antibody sandwich structure. The test was started by loading the mixture of sample, IFNs and 1% BSA-PBS onto the sample pad, and all liquid was absorbed and migrated along the strip by capillary forces.16 The IFNs/CRP complexes then were captured by the MC01 antibodies on the NC membrane via antigen-antibody interaction between CRP and MC01 antibody, and the signal can be read with a portable ultraviolet lamp due to the robust fluorescence of IFNs. As the liquid continued migrating, free IFNs moved further and were trapped by the goat anti-mouse IgG to form a control zone. If no CRP was present in the sample, the IFNs should flow past the test zone without interaction and only react with the goat anti-mouse IgG on the control zone. The determination of the results was illustrated in Figure 3C. The sample was considered positive when two fluorescence lines appeared on the test zone and control zone within 20 min (Figure 3C1), and negative when only the control line was seen (Figure 3C2). An invalid test was the one with only a test line or no lines (Figure 3C3 and 3C4). Figure 3C5 showed the picture of a positive result, and the test line and control line can be seen clearly with a portable ultraviolet lamp. Sensitivity of the lateral flow test strip assay for quantification of CRP
Under optimized detection conditions (details in Supporting Information S3), the sensitivity of the IFN-based lateral flow test strip assay was investigated. Firstly, 1 μL of sample (CRP standard sample) and 0.15 nM IFNs were mixed with 1% BSA-PBS in a well of 96-well plate (the final volume was 80 μL). Then the solution containing CRP at different concentration, such as 0.178 nM, 0.714 nM, 2.86 nM, 5.71 nM and 11.4 nM, respectively, was loaded onto the sample pad of the lateral flow test strip. As expected, only control line was observed in the absence of CRP. In contrast, two lines were observed in the presence of CRP. Meanwhile, the brightness of the test line increased with the increasing concentration of CRP (Figure 4A). This test can be qualitative by estimating the brightness and also quantitative by measuring the fluorescence intensity of the test line. As shown in Figure 4B, a linear relationship was obtained in a wide range of 0.178 nM to 11.4 nM with a linear correlation coefficient (R 2) of 0.995. The detection limit was determined to be 27.8 pM (equivalent to 3.89 ng/mL). For the sample 80 folds diluted, the linearity range of 0.178 nM to 11.4 nM was equivalent to 2.0 mg/L-128.0 mg/L. At the same time, the detection limit of 3.89 ng/mL was equivalent to 0.311 mg/L. For the sample, the detection limit was one thirtieth of the clinical threshold value (10 mg/L). Such high sensitivity can predict cardiovascular risk. Individuals are typically stratified into three groups of low cardiovascular risk (3.0 mg/L). Furthermore, it can be used to accurate diagnosis of bacterial (>75.0 mg/L) or viral infection (10.0 mg/L to 20.0 mg/L) and cancer (10.0 mg/L to 40.0 mg/L). 43,44 Compared 4
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Analytical Chemistry
with conventional Au-based lateral flow test strip for CRP detection, the IFN-based lateral flow strip can increase sensitivity by 257
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folds.45 It was attributed to the use of FNs, which contained 332 ± 8 QDs in one FN. They not only inherited the unique optical properties of QDs, but also 380 folds higher than QDs in brightness. Hence, compared with other methods for CRP detection, a higher sensitive and timesaving assay was obtained (Table 1),9,45-48 which indicated that the IFN-based lateral flow have a very promising prospect in the high sensitive POC detection of protein biomarker. All these experimental results proved that IFNs were able to be utilized in the lateral flow test strip platform as an excellent fluorescent reporter, establishing a sound basis for rapid POC quantitative detection for protein biomarker. Robustness of the IFN-based lateral flow test strip assay for clinical application. To investigate the feasibility of IFN-based lateral flow test strip assay for clinical application, the specificity was also tested by employing several types of protein (12.5 μg/mL PSA, 20.4 μg/mL CEA, 5.2 μg/mL AFP and 12.5 μg/mL HSA) as negative controls. The results showed in Figure 5A, the fluorescence intensity of CRP (25.0 ng/mL) sample was conspicuously higher than negative control ones. We calculated that the fluorescence intensity for CRP sample generally was 10 folds than that for those nonspecific protein samples even when their concentration was nearly 200-800 folds than that of the CRP sample. Besides, no obvious signal was found in the controls even when the concentration of interference protein (CEA) was about 800 folds of CRP sample, suggesting the high specificity of the method. It also indicated the high sensitivity and strong anti-interference of the IFNs. Hence, IFNs were able to be utilized in complex matrix directly for quantitative detection of protein biomarker. In corroboration of the feasibility of the IFN-based lateral flow test strip for clinical application, the assay was applied to FBS samples. As expected, the results were similar with the detection performance in buffer (Figure 5B). Meanwhile, a good calibration curve was obtained with the detection limit of 34.8 pM (Figure 5C). It was slightly different with the detection limit of 27.8 pM in buffer. This also indicated that complex matrix had no significant effect on the binding between IFNs and CRP. Furthermore, it provided the strong evidence to prove the high anti-interference of the IFNs.
Good precision and reproducibility are of great importance for further application of the method. To evaluate the precision and reproducibility of the method, the intra-assay and inter-assay were executed using 0.178 nM, 2.85 nM and 11.4 nM CRP standard samples. According to the data showed in Table 2, intra-assay CV and inter-assay CV of the method were calculated to be 5.3% and 6.6%, respectively, which confirmed the high reproducibility and good precision of the assay. It was presumably due to the highly uniform particle size and stability of the FNs. As we known, for high performance in lateral flow test strip platform, reporters must be narrow size distributions, large surface areas (for maximal protein or enzyme binding), high stability to provide robust signal and good dispersion in liquid media. The FNs were highly uniform in particle size distribution that were proved in Figure 1. As shown in Figure 6A, the fluorescence intensity of the FNs essentially kept stability with increasing storage time, and it can be seen that IFNs could retain their bioactivity when used in test strips even after 6 months' storage (Figure 6B). Hence, the reproducibility of IFN-based test strip was better in comparison to other reporter-based lateral flow test strip (Table S2, Supporting Information). 23, 49 Taken together, these above results indicated that the detection assay possessed good reproducibility, high stability, and strong anti-interference ability and could be applied in complex matrix. Detection of CRP in peripheral blood plasma samples from cancer patients. Having proved that the CRP could be detected with IFN-based test strip sensitively, rapidly, specifically and quantitatively, the method was applied to the detection of CRP in the peripheral blood plasma samples from twenty-nine cancer patients (including lung and breast cancer patients) and five healthy people. The results were shown in Figure 7. According to the results, for healthy humans, blood plasma level of CRP was lower than 10 mg/L, and it elevated in lung cancer patients but not in breast cancer patients. This may because elevated CRP was a result of an underlying lung cancer or a premalignant state. Another possible reason was that elevated CRP is associated with lung cancer carcinogenesis.45,50 Therefore, these results indicated that the IFNs coupled with lateral flow test strip platform may bring a tremendous infusion of promise to the rapid quantitative detection of CRP in clinical 5
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applications.
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Conclusions In summary, FN as a promising alternative reporter was integrated with lateral flow test strip platform, which was able to detect CRP with a detection limit of 27.8 pM in buffer and 34.8 pM in serum. Such high sensitivity enabled the accurate diagnosis of bacterial or viral infection and several types of cancer, and can be used to predict cardiovascular risk. The assay can be completed within 20 min with high sensitivity, high specificity, and good reproducibility (intra-assay CV and inter-assay CV
