Development of a SERS-Based Rapid Vertical Flow Assay for Point-of

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)

Letter

Development of a SERS-based rapid vertical flow assay for point-of-care diagnostics Osai J.R. Clarke, Barbara I. Goodall, Hok Ping Hui, Neeraj Vats, and Christa L. Brosseau Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04710 • Publication Date (Web): 17 Jan 2017 Downloaded from http://pubs.acs.org on January 17, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Development of a SERS-based rapid vertical flow assay for point-of-care diagnostics O.J.R. Clarkea, B.L. Goodallb, H.P. Huib, N. Vatsb, C.L. Brosseaua*

Abstract Point-of-care (POC) diagnostic testing platforms are a growing sector of the healthcare industry as they offer the advantages of rapid provision of results, ease of use, reduced cost, and the ability to link patients to care. While many POC tests are based on chromatographic flow assay technology, this technology suffers from a lack of sensitivity along with limited capacity for multiplexing and quantitative analysis. Several recent reports have begun to investigate the feasibility of coupling chromatographic flow platforms to more advanced read-out technologies which in turn enable on-site acquisition, storage and transmission of important healthcare metrics. One such technology being explored is surface-enhanced Raman spectroscopy, or SERS. In this work, SERS is coupled for the first time to a rapid vertical flow (RVF) immunotechnology for detection of anti-HCV antibodies in an effort to extend the capabilities of this commercially available diagnostic platform. High quality and reproducible SERS spectra were obtained using reporter-modified gold nanoparticles (AuNPs).

Serial dilution studies

indicate that the coupling of SERS with RVF technology shows enormous potential for nextgeneration POC diagnostics. * To whom correspondence should be addressed: Christa L. Brosseau ([email protected]) Phone (902) 496-8175 Fax (902) 496-8104 Department of Chemistry, Saint Mary’s University, Halifax, Nova Scotia, Canada, B3H 3C3. a. Department of Chemistry, Saint Mary’s University, Halifax, Nova Scotia, Canada. b. MedMira Laboratories Inc. Halifax, Nova Scotia, Canada. KEY WORDS: Surface-enhanced Raman spectroscopy, rapid vertical flow assay, gold, point of care, diagnostics, immunoassay, hepatitis C, disruptive technology, nitrocellulose, protein A

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Introduction Point-of-care (POC) diagnostic tests are becoming exceedingly important tools for health care providers, patients, and individuals. POC tests can aid in the diagnosis of disease, or monitor health, and are employed in numerous settings offering several advantages including ease of use, rapid analysis, and in-home health care monitoring.1-3 One of the most widely used POC platforms is the lateral flow immunoassay (LFIA). Due to the very specific nature of the antigen-antibody interactions, LFIA can monitor analytes in complex matrices such as urine or whole blood.4,5 Despite the numerous advantages of LFIAs, several limitations remain including lack of quantitation capabilities and limited sensitivity compared to laboratory based assays.6,7 Recent work has highlighted some improvements to LFIA technology in terms of both improved visual labels8-10 as well as new detection strategies11,12; however, despite these improvements LFIA continues to suffer from poor precision and limited sensitivity. An alternative to LFIA are rapid vertical flow assays (RVF) which offer several key advantages, including faster analysis time, no timing requirement (results available immediately after application since there is no lateral flow of solution) and the absence of a false-negative inducing hook effect.13 As a result, many next generation POC diagnostics are beginning to explore RVF as opposed to LFIA.14-16 In an effort to address the limitations of POC technologies, several groups have explored the coupling of various detection modalities to conventional flow assay platforms. For example, reflectance spectroscopy17, fluorescence spectroscopy18 and electrochemical signal generation19 have all been explored in this regard. Over the past five years, surface-enhanced Raman spectroscopy, or SERS, has emerged as a promising detection modality for coupling to lateral flow immunoassays due primarily to the promise of unparalleled sensitivity. For example, Wang


Page 2 of 20

Page 3 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


et al. reported the use of a Raman label bound to an antibody-functionalized gold nanoparticle which allowed for the indirect detection of rabbit IgG.20 Recent reports have extended this concept to the quantitation of HIV-121 and staphylococcal enterotoxin B22, with reported SERSbased limits of detection of 0.24 pg/mL and 1 pg/mL, respectively. In the work presented herein, we seek to explore the extent to which SERS-based detection can be extended to rapid vertical flow (RVF) immunoassay technology in order to address the current limitations of poor sensitivity and lack of quantitation. Detection of Hepatitis C (HCV) biomarkers was used as a model system. There are a multitude of factors which put people at risk for HCV, some risks are related to lifecycle, others due to date of birth, as well as place of birth. The US CDC recommends that persons born from 1945-1965 be screened for HCV at least once. With the need to screen all persons at risk for HCV so that those infected can be treated and cured, rapid POC tests for HCV have become important tools to facilitate initial diagnosis where no symptoms are typically present. The SERS-based RVF platform in this work utilized the interaction between antigens derived from conserved regions of hepatitis C viral proteins, spotted onto the test membrane, and anti-HCV antibodies that would be in an infected patient’s blood sample. The resultant immune complexes were detected using a direct label incorporating a Raman active marker. In this research, we present the first example of SERS coupled to a rapid vertical flow (RVF) immunoassay. This platform represents a threedimensional SERS substrate which can be used for the future development of a quantitative immunoassay.

Experimental Reagents, Solutions and Materials



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

para-aminothiophenol (p-ATP, 97%) was used as the Raman reporter molecule for this work, and was purchased from Sigma Aldrich. Protein A from Staphylococcus aureus (salt-free, lyophilized powder) and bovine serum albumin (lyophilized powder, ≥96%) were also purchased from Sigma Aldrich. Gold chloride (ACS reagent, ≥49% Au basis, Sigma Aldrich), sodium citrate (ACS reagent, ≥99.0%, Sigma Aldrich), sodium carbonate (ACS reagent, ≥99.5%, Sigma Aldrich) and sodium bicarbonate (ACS reagent, ≥99.7%, Sigma Aldrich) were used in the preparation of the Au colloids used in this work. All solutions were prepared using Millipore water (≥18.2 MΩ cm). Miriad™ HCV/HIV Rapid Vertical Flow (RVF) assays multiplexed for hepatitis C virus (HCV) and human immunodeficiency virus 1/2 (HIV) were supplied by MedMira Laboratories Inc. for testing (details of the assay are provided in the supporting information). A monoclonal antibody specific for HCV antigens was dialyzed against PBS at a concentration of 3.4 mg/mL and supplied by Cedarlane. All glassware for this research was cleaned by immersion in neat sulfuric acid overnight, followed by careful rinsing with Millipore water. Preparation of Au nanoparticles and surface functionalization ~13 nm gold nanoparticles (AuNPs) were prepared using a standard method from the literature.23 Details of the NP synthesis are provided in the supporting information. Once the gold nanoparticles were prepared, 10 µL of a 1.0 mg/mL protein A solution prepared in water was added to 990 µL of the AuNP colloidal solution in an Eppendorf tube. The tube was then placed on an orbital shaker platform for 30 minutes at room temperature. Next, 390 µL of 0.08 mM pATP was added to the tube containing the protein A-modified AuNPs, and gently aspirated several times with the pipette tip followed by the addition of 1.0 µL of 1.0% v/v bovine serum albumin. The tube was then placed back on the orbital shaker platform for an additional 30 minutes. Once complete, the sample was centrifuged at 15,000 rpm for 30 minutes (Labnet


Page 4 of 20

Page 5 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


PRISM microcentrifuge, Edison, NJ, USA). The supernatant was removed, and the pellet was reconstituted with 100 µL of capping buffer, provided by Medmira. The capping buffer represents a proprietary blend, but in general was composed of PBS saline, synthetic polymers and anti-microbial agents.

Instrumentation Raman and SERS experiments for the developed RVF test cartridge were conducted using a DeltaNu benchtop Raman spectrometer equipped with a 785 nm laser (Intevac Photonics, Santa Clara, USA).

The instrumental set-up is pictured in Figure S-1. The spectrometer

resolution is 5 cm-1 and it is equipped with an air-cooled CCD detector and a right angle optics attachment. Sample acquisition time was 30-60 seconds at laser powers ranging between 10.655.9 mW. All Raman data is corrected for both laser power and acquisition time. Origin 8.1 was used for the spectral processing and data analysis (OriginLab Corporation, Northampton, MA, USA). Instrumentation used for characterization of the AuNPs as well as the nitrocellulose membrane is outlined in the supporting information. Results and Discussion Characterization of RVF test cartridge Figure S-2 shows the SEM image of the nitrocellulose test cartridge membrane used in this study. The porous nitrocellulose structure is evident, with pore diameters ranging from a few hundred nanometers to several microns. An important consideration for this work was to what extent the normal Raman signal for the nitrocellulose (NC) polymer would present a background interference. Figure S-3 shows the normal Raman signal for the bare nitrocellulose membrane, recorded for five different spots on the same membrane surface. Of particular note are several



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

strong Raman bands at 856 cm-1, 1288 cm-1 and 1379 cm-1 which correspond to the ν(NO), νs(NO2) and δ(C-H) modes of nitrocellulose, respectively.24 In addition, peaks between ~1050 cm-1 and ~1150 cm-1 due to ν(C-O) for the pyranose sugars are also observed to be present.24 In recent years, nitrocellulose has been increasingly explored as a substrate for SERS, due in large part to its heavy usage in lateral flow assays.25,26

Characterization of AuNPs The initial characterization of the AuNP colloidal sol was completed by measuring the extinction spectrum using a UV-vis-NIR spectrophotometer. A representative spectrum is plotted in Figure 1A. The extinction maximum was observed to be at 520 nm, which is consistent with spherical AuNPs.27 In addition, the band was observed to be fairly narrow, an indication that the nanoparticles were fairly monodisperse. This observation was then further evaluated using transmission electron microscopy. Figure 1C shows a representative TEM image of the bare AuNPs prepared in this study, along with a histogram (Figure 1B) showing the size dispersion of the nanoparticles. With an average diameter of 12.6 nm (±1.3 nm), the gold nanoparticles are fairly monodisperse and are of an appropriate size to support the localized surface plasmon resonance required for efficient SERS enhancement.27 Evaluation of SERS performance for RVF test The first consideration for this project was the appropriate selection of a Raman reporter molecule. As the nitrocellulose membrane has a strong Raman signature itself, it was important to choose a reporter molecule that would not only be strongly bound to the AuNP surface, but which would also have a signal with peaks not overlapping that of the nitrocellulose. As a result, para-aminothiophenol (p-ATP) was chosen for this work. Figure S-4 provides an overlay of the


Page 6 of 20

Page 7 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


normal Raman signal for the nitrocellulose with the SERS signal for p-ATP. The predominant pATP peaks at ~1080 cm-1 (v(C-S)) and ~1590 cm-1 (v(C-C)) are excellent marker peaks which do not interfere with the membrane signal.28,29 The next step in the project was to evaluate the SERS performance for the developed test spot. When the AuNPs were functionalized with p-ATP according to the procedure outlined above, a colour change was noted from red to purple after the centrifugation step, as can be seen in Figure 2. When the modified AuNPs were then used for the test, the developed spot was also purple, as opposed to red (Figure 2). This is important as it indicates that the AuNPs, once modified with the Raman reporter, maintain their functionality for the immunoassay. This redshifted colour change is an indication that upon modification with p-ATP, the AuNPs are likely slightly aggregated and / or have undergone a shape change that is causing the LSPR to shift. Studies with 30 nm AuNPs (data not shown) have indicated that upon modification with protein A and p-ATP, there is a relative increase in the number of hexagonal and triangular nanoparticles, as well as enhanced aggregation. In terms of SERS enhancement, this is an important finding as nanoparticle aggregation is well known to be a requirement for obtaining high quality SERS signals. In fact, we have observed that if there is no color change from red to purple after the conjugation steps, the particles will not be SERS-active. Figure 3 shows the SEM image of the developed test spot after the test procedure was completed with the HCV monoclonal antibody and InstantGold Cap. The AuNPs can clearly be observed on the surface of the membrane and percolating down through the membrane. When the membrane was removed and prepared in cross-section, the AuNPs could still be observed in the deep interior of the membrane. This is an important finding as it suggests that this AuNPdecorated nitrocellulose membrane is functioning as a 3-dimensional SERS substrate, where a



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

three dimensional focal volume is accessible for SERS signal acquisition, as opposed to a 2dimensional focal area, as is observed for traditional non-porous SERS substrates. This observation is further supported by Figure S-5, where the SERS signal is recorded for the developed test spot prior to the addition of buffer, and after each subsequent buffer drop. With each successive drop of buffer, no signal loss is observed, indicating that the signal is not attenuated by dilution, and the SERS signal is in fact coming from a focal volume. Using a scanning knife edge measurement and measuring the intense Si-O vibrational mode for silicon, the focal depth was determined to be ~1 mm. With a laser beam diameter of 100 µm, a laser spot diameter at focus of 25 µm, and assuming a focal volume approximated as a truncated cone, this corresponds to a focal volume of ~3.4 x 106 µm3. Recent reports have begun to highlight the benefit offered by such 3-D SERS substrates. The first examples of truly 3-D SERS substrates were those prepared using paper30 and fabric31 as the substrate, in conjunction with colloidal Au or Ag. During preparation of these substrates, the colloidal sol percolated down through the layers of the porous substrate, thus representing a SERS substrate in three dimensions. Such 3-D SERS substrates have recently gained substantial popularity for a variety of sensing applications.32-35 Figure 4 shows the SERS spectra collected for 10 different spots on the developed test spot. This measurement simply required movement of the test cartridge by 1 mm in order to access a new area, followed by signal collection. Since the laser spot diameter is only ~100 µm, many different areas within the developed test spot can be probed. In the future, this quality should lend itself well to multiplexed analysis. The SERS signal for the Raman reporter is very good, especially considering that the size of the nanoparticles is not optimal for efficient SERS enhancement, and no significant interference from the nitrocellulose membrane was observed for


Page 8 of 20

Page 9 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the main Raman reporter peaks at ~1080 and ~1590 cm-1. Signal for the nitrocellulose membrane is continually present, especially for the peaks at 856 cm-1 and 1288 cm-1. In addition, the membrane peaks are remarkably reproducible, and as such may be useful for internal standardization of the SERS signal, as reported by Bishnoi et al.25 An important observation that can be made at this point is the signal uniformity observed for the SERS signal. In general, SERS suffers from a lack of signal uniformity due to the non-uniform arrangement of SERS-active hot spots on the SERS substrate; this issue has been well documented in the literature.36,37 This lack of signal homogeneity is problematic for quantitative SERS analysis, and has limited the success of SERS for applications which require a quantitative result. For the present study, the coefficient of variation (CV) was determined for the p-ATP peak at 1083 cm-1, normalized to the intensity of the NC membrane peak at 1290 cm-1 (details provided in supporting information), and was found to be 16%.25 The coefficient of variation is a reliable way of reporting the variation in SERS intensity, as it relates the signal intensity to the signal variability and both of these parameters are of significant importance in the development of SERS-based sensor technology. This is important since a future goal of this platform is the enabling of quantitative analysis from the RVF assay. It is quite likely in this case that by using the NC membrane as the SERS substrate, which offers the advantage of acting as a 3-D SERS platform, a greater signal homogeneity is obtainable since a focal volume is probed as opposed to a 2-D focal area. While the SERS signal observed for the undiluted antibody presented above was indeed very promising, a central goal of this work was to evaluate the usefulness of SERS for extending the limit of detection for the RVF platform beyond what is possible by visual determination, and in turn improving the sensitivity of the test. Figure 5 shows the SERS signal obtained for a serial dilution series of the monoclonal antibody, and the corresponding test cartridge images are



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 20

provided in Figure S-6. As can be seen from Figure 5, SERS signal is readily observed down to 1/64 dilution, which corresponds to an antibody concentration of 53.1 µg mL-1. This is also the limit of detection observed by eye for the visual test with these modified AuNPs. Since the focus of this research was simply to illustrate that efficient coupling of RVF technology and SERS is possible for diagnostic assay applications, future work on this topic will focus on optimizing the SERS parameters (nanoparticle size and shape, surface functionalization strategy, etc) such that much improved limits of detection can be obtained. Conclusions In conclusion, this work represents the first time SERS has been coupled with rapid vertical flow immunoassay technology. It was shown that the nitrocellulose membrane functions as an excellent SERS substrate once the modified Au colloids have been deposited onto the test strip, and the vertical flow allows for enhanced loading of the substrate with AuNPs. The SERS signals obtained for p-ATP, the Raman reporter used in this work were found to be intense and highly reproducible, with very little spot-to-spot variation in signal.

Future work will seek to

optimize the coupling of SERS and RVF technology in an effort to create a truly quantitative POC platform for the multiplexed detection of disease from whole blood. Optimization strategies to improve the limit of detection will include increasing the size of the AuNPs to allow for better SERS enhancement, improving the surface modification with the Raman reporter and exploring alternate Raman reporters. In addition, future studies will seek to illustrate quantitative detection in whole blood. Acknowledgements This work was supported by the National Research Council Industrial Research Assistance Program (NRC-IRAP). C.L. Brosseau also acknowledges support from the Natural


Page 11 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Sciences and Engineering Research Council (NSERC) Discovery Grant program; in addition the Canadian Foundation for Innovation (CFI) and the Nova Scotia Research and Innovation Trust (NSRIT) provided infrastructure support for this work. We are also grateful to Dr. Xiang Yang and Dr. Ping Li for assistance with the SEM and TEM imaging, respectively. Conflict Of Interest The authors declare the following competing financial interest(s): B.L. Goodall, H.P. Hui, and N. Vats are employees of MedMira Laboratories. H.P Hui is the co-founder MedMira Laboratories, Inc. All remaining contributing authors declare no competing financial interests. References (1) Yager, P.; Domingo, G.J.; Gerdes, J. Annu. Rev. Biomed, Eng. 2008, 10, 107-144. (2)Gubala, V.; Harris, L.F.; Ricco, A.J.; Tan, M.X.; Williams, D.E. Anal. Chem. 2012, 84, 487515 (3) Warsinke, A. Anal. Bioanal. Chem. 2009, 393, 1393-1405. (4) Lawn, S.D.; Dheda, K.; Kerhoff, A.D.; Peter, J.G.; Dorman, S.; Boehme, C.C.; Nicol, M.P. BMC Infectious Diseases. 2013, 13, 407-416 (5) Corstjens, P.L.A.M.; Tjon Kon Fat, E.M.; de Dood, C.J.; van der Ploeg-van Schip, J.J. Franken, K.L.M.C; Chegou, N.N.; Sutherland, J.S.; Howe, R.; Mihret, A.; Kassa, D.; van der Vyver, M.; Sheehama, J.; Simukonda F.; Mayanja-Kizza, H.; Ottenhoff, T.H.M.; Walzul, G. Geluk, A. Clinical Biochemistry. 2016, 29, 22-31. (6) Posthuma-Trumpie, G.A.; Korf, J.; van Amerongen, A. Anal. Bioanal. Chem. 2009, 393, 569-582. (7) Sajid. M.; Kawade, A-N.; Daud, M. Journal of Saudi Chemical Society. 2015, 19, 689-705. (8) Song, C.; Liu, J.; Li, J.; Liu, Q. Biosensors and Bioelectronics. 2016, 85, 734-739. (9) Chen, Y. Sun, J.; Xianyu, Y.; Yin, B.; Niu, Y.; Wang, S.; Cao, F.; Zhang, X.; Wang, Y.; Jiang, X. Nanoscale. 2016, 8, 15205-15212. (10) Zhang, K.; Wu, J.; Li, Y.; Wu, Y.; Huang, T.; Tang, D. Microchim. Acta. 2014, 181, 14471454.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(11) Fernández-Sánchez, C.; Gallardo-Soto, A.M.; Rawson, K.; Nilsson, O.; McNeil, C.J. Electrochemistry Communications. 2004, 6, 138-143. (12) Li, Z.; Wang, Y.; Wang, J.; Tang, Z.; Pounds, J.G.; Lin, Y. Anal. Chem. 2010, 82, 70087014. (13) Cytodiagnostics Vertical Flow Immunoassays. http://www.cytodiagnostics.com/store/pc/Vertical-Flow-Immunoassays-d38.htm (accessed Nov 3, 2016). (14) Owen, S.M.; Spira, Y.T.; Ou, C.Y.; Pau, C.P.; Parekh, B.S.; Candal, D; Kuehl, D.; Kennedy, M.S.; Rudolph, D.; Luo, W. Dealtorre, N.; Masciotra, S.; Kalish, M.L.; Cowart, F. Barnett, T.; Lal, R.; McDougla, J.S. Journal of Clinical Microbiology. 2008, 46, 5, 1588-1595. (15) Chinnasamy, T.; Segerink, L.I.; Gantelius, J.; Andersson Svahan, H. Clin. Chem. 2014, 60 9, 1209-1216. (16) Oh, Y.K.; Joung, H.A.; Kim, S.; Kim, M.G. Lab Chip. 2013, 13, 5, 768-772. (17) Wittmann, C.; Schreiter, P-Y. J. Agric. Food Chem. 1999, 47, 2733-2737. (18) Kato, N.; Caruso, F. J. Phys. Chem. B. 2005, 109, 19604-19612. (19) Tang, D.; Yuan, R.; Chai, Y. Anal. Chem. 2008, 80, 1582-1588. (20) Wang, G.; Driskell, J.D.; Hill, A.A.; Dufek, E.J.; Lipert, R.J.; Porter, M.D. Annu. Rev. Anal. Chem. 2010, 3, 387-407. (21) Fu, X.; Cheng, Z.; Yu, J.; Choo, P.; Chen, L.; Choo, J. Biosensors and Bioelectronics. 2016, 78, 530-537. (22) Hwang, J.; Lee, S.; Choo, J. Nanoscale. 2016, 8, 11418-11425. (23) Hayat, M.A. (Ed.) Colloidal Gold: Principles, Methods, and Applications. Vol 1. Academic Press, Inc. San Diego: California, 1989. (24) Moore, D.S.; McGrane, S.D. Journal of Molecular Structure. 2003, 661-612, 561-566. (25) Bishnoi, S.W.; Lin, Y. Tibudan, M.; Huang, Y.; Nakaema, M.; Swarup, V.; Keiderling, T.A. Anal. Chem. 2011, 83, 4053-4060. (26) Zhang, P.; Zhang, R.; Gao, M.; Zhang, X. Appl. Mater. Interfaces. 2013, 6, 370-376. (27) Joseph, V.; Matschulat, A.; Polte, J,; Rolf, S.; Emmerling, F.; Kneipp, J. J. Raman Spectrosc. 2011, 42, 1736-1742. (28) Osawa, M.; Matsuda, N.; Yoshii, K.; Uchida, I. J. Phys. Chem. 1994, 98, 12702-12707.


Page 12 of 20

Page 13 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(29) Huang, Y-F.; Zhu, H-P.; Liu, G-K, Wu, D-Y.; Ren, B.; Tian, Z-Q. J. Am. Chem. Soci. 2010, 132, 9244-9246. (30) Hoppmann, E.P.; Yu, W.W.; White, I.M. Methods. 2013, 63, 219-224. (31) Robinson, A.M.; Zhao, L.; Shah ALam, M. Y.; Bhandari, P.; Harroun, S.G.; Dendukuri, D.; Blackburn, J. Brosseau, C.L. Analyst. 2015, 140, 779-785. (32) Liu, J.; Zhou, J.; Tang, B.; Zeng, T.; Li, Y.; Li, J.; Ye, Y. Applied Surface Science. 2016, 386, 296-302. (33) Liu, H.; Yang, L.; Liu, J. Trends in Analytical Chemistry. 2016, 80, 364-372. (34) Liu, H.; Yang, Z.; Meng, L.; Sun, Y.; Wang, J.; Yang, L.; Liu, J. Tian, Z. J. Am. Chem. Soci. 2014, 136, 5332-5341. (35) Kurouski, D.; Large, N.; Chiang, N.; Greeneitch, N.; Carron, K.T.; Seideman, T.; Schatz, G.C. Van Duyne, R.P. Analyst. 2016, 141, 1779-1788. (36) Sackmann, M.; Materny, A. J. Raman Spectrosc. 2006, 37, 305-310. (37) März, A.; Ackermann, K.R.; Malsch, D.; Bocklitzz, T.; Henkel, T. Popp, J. J. Biophoton. 2009, 2, 4, 232-242.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 20

Figure Captions:

Figure 1. (A) Extinction spectrum of the bare colloidal AuNPs (B) size distribution for the AuNPs as imaged by TEM (C) TEM image of the bare gold nanoparticles Figure 2. (A) The suspension of protein A conjugated AuNPs exhibited a red color, and results in a red developed test spot (B) The suspension of protein A and p-ATP conjugated AuNPs exhibited a purple color, resulting in a purple developed test spot. Figure 3. SEM images of the test cartridge membrane after a positive specimen was introduced. (A) the surface of the HCV test dot (B) the cross section of the HCV test dot. Figure 4. SERS spectra collected for 10 different spots on the developed test spot for undiluted monoclonal antibody. Laser excitation was 785 nm. Laser power at the membrane was 55.9 mW, and acquisition time was 60 s. Figure 5. SERS signal obtained for a serial dilution series of the monoclonal antibody. Laser excitation was 785 nm. Laser power at the membrane was 55.9 mW, and acquisition time was 60 s. “0” represents the negative control study where no monoclonal antibody was introduced, but the test was developed and analyzed using the same protocol.


Page 15 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figures

Figure 1



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2


Page 16 of 20

Page 17 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4


Page 18 of 20

Page 19 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TOC Figure


Page 20 of 20

Development of a SERS-Based Rapid Vertical Flow Assay for Point-of

Recommend Documents