Multiplex Analysis on a Single Porous Hydrogel Bead with Encoded

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Multiplex analysis on a single porous hydrogel bead with encoded SERS nanotags Bing Liu, Di Zhang, Haibin Ni, Delong Wang, Liyong Jiang, Degang Fu, Xiaofeng Han, Chi Zhang, Hong-Yuan Chen, Zhongze Gu, and Xiang-Wei Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b14942 • Publication Date (Web): 18 Dec 2017 Downloaded from http://pubs.acs.org on December 19, 2017

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Multiplex analysis on a single porous hydrogel bead with encoded SERS nanotags ∥

Bing Liu,†,‡,§ Di Zhang,†,‡ Haibin Ni,†,‡ Delong Wang,†,‡ Liyong Jiang, Degang Fu,†,‡ Xiaofeng Han,†,‡,§ Chi Zhang, ⊥ Hongyuan Chen,# Zhongze Gu†,‡ and Xiangwei Zhao*†,‡,§

†

State Key Laboratory of Bioelectronics, School of Biological Science and Medical

Engineering,

‡

National Demonstration Center for Experimental Biomedical

Engineering Education, §Key Laboratory of Environmental Medicine Engineering of Ministry of Education, Southeast University, Nanjing 210009, China ∥

Department of Physics, School of Science, Nanjing University of Science and

Technology, Nanjing 210094, China ⊥

#

Nanjing Institute of Product Quality Inspection, Nanjing 210019, China

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry

and Chemical Engineering, Nanjing University, Nanjing 210093, China

ABSTRACT: Bead-based assays have drawn more and more attentions in biomedical fields. Herein, we proposed a novel approach to achieve multiplex analysis on a single porous hydrogel bead (PHB) with Raman dyes (RDs) encoded core-shell surface-enhanced Raman scattering (SERS) nanotags. Owing to the amplified signal RDs by core-shell metal structure of the nanotag and the high surface area to volume ratio (SVR) of the PHB, the sensitivity and linear dynamic range (LDR) of the


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as-proposed multiplex analysis method are significantly improved. We anticipate this approach to be widely used in the multiplex assays and biosensors. KEYWORDS: bead-based assay, multiplex assay, porous hydrogel bead, core-shell SERS nanotags, linear dynamic range

Bead-based assays have been widely used in disease diagnostic, biological, and chemical analysis because of their high flexibility in usage.1-3 For example, they can be integrated with microfluidic chips, optical fibers, micro-wells, and tips.4-7 The surface of bead can be easily functionalized with various biorecognition receptors, such as antibody, molecular imprint, aptamer, and peptide. Therefore, the bead can act as solid phase carrier in assays or sensors.8,9 Compared with conventional planar carrier like micro-well or microarray commonly used in ELISA (Enzyme-linked immunosorbent assay), the bead with high surface area to volume ratio (SVR) can be effectively mixed with samples by vortex, which leads to higher sensitivity and shorter reaction time.10,11 In addition, over the past decades, many considerable encoding techniques have been developed, and the beads with unique codes can form suspension array, by which high throughput and multiplex assays with faster detection and less sample are realized. However, an encoded single bead only bears one kind of molecular probe on its surface, posing a challenge for further sample reduction and higher throughput. For this reason, recently some attempts have been made with orthogonal chemistry or microfabrication to obtain multiple sensing elements on a single particle.12-14 However, there are still limitations in terms of reactive probes number on particle surface or the fabrication cost. In addition, the sensitivity and



linear dynamic range (LDR) may be constrained in the multiplex analysis on a single particle due to the reduced reactive surface area of one probe, which is undesirable for the high throughput screening of biomolecules in wide ranges of concentration.15,16 Hence, in this paper, we proposed a novel approach to achieve multiplex protein biomarkers analysis on a single porous hydrogel bead (PHB) with multiplex antibody probes modified. The biomarkers could be deciphered by Raman dyes (RDs) encoded SERS (Surface-enhanced Raman scattering) nanotags. As illustrated in Figure 1, first, a single PHB of polyacrylamide (PAM) was made from self-assembled colloidal crystal template and immobilized with multiplex capture antibodies. Then, it reacted with protein biomarkers and detection antibodies labeled with encoded SERS nanotags successively. Finally, Raman spectrum was measured and deciphered to get the encoding Raman shift peaks of RDs and the results of multiplex detection.17 As a proof of concept, we used the as-proposed bioassay for the analysis of AFP (Alpha-fetoprotein) and CEA (Carcinoembryonic antigen), which are two commonly used biomarkers for the clinical diagnosis of a series of tumors like liver and colon.18,19 In this method, the high SVR of the porous hydrogel materials makes it possible to increase the density of the binding biomarkers and enlarge the concentration range to be detected. This is especially valuable for the high throughput screening of practical samples, where protein concentrations may range from fg mL-1 to µg mL-1.15 Wide linear dynamic range means dilutions of the sample will be avoided for quantitative analysis and the cost and time will be saved.15 On the other hand, the sensitivity of the


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system depends mostly on the signal to noise ratio (SNR) could not be decreased. Here, Raman dyes were embedded in the interior gap between gold core and silver shell, and the resultant SERS nanotags were used as labels instead of fluorescence dyes (Figure 1). Owing to the high efficient plasmon enhanced Raman scattering of the gap, Raman signal of the RDs is amplified more than 8 orders of magnitude, which makes it possible to detect one single nanotag.20,21 Except for that, core-shell nanostructure-based SERS nanotags with highly uniform and reproducible signals are also important for ultrasensitive detection of biomarkers.22 In addition, Raman dyes with different Raman spectral features are used as encoding elements of the SERS nanotags, and they could be decoded by one shot of Raman excitation, which brings multiplex detection on a single bead into practice. This new technology holds great promise for applications in the discovery or high throughput quantitative analysis of proteins in many fields like precision medicine, system biology and so on.

Figure 1. Schematic diagram of multiplex protein biomarkers detection on a single PHB by SERS nanotags. Inset is schematic structure of SERS nanotag, Raman dyes are embedded into the interior gap of Au core and Ag shell. High sensitivity and wide linear dynamic range are desired for quantitative analysis.



However, they usually could not be achieved simultaneously at the interfaces of flat solid and liquid.16 One of the main reasons lies in the low SVR of the solid phase. Therefore, in this work, we fabricated an inverse photonic crystal bead with PAM hydrogel by the template of self-assembled silica colloidal crystal bead (CCB) to increase the surface area of photonic crystal bead. The PAM pregel solution was first infiltrated into the interval of 236 nm silica nanoparticles in a CCB and then cured with UV light. After the silica nanoparticles in the hydrogel were removed with hydrofluoric acid etching, a PHB was obtained, as shown in Figure 2a and 2b. Since the silica nanoparticles are monodispersed and connected in the template, the pores are ordered and connected throughout the hydrogel. The diameters of the opening windows on the surface and the interconnecting apertures between adjacent pores are close to 236 and 100 nm, respectively. The binding kinetics of the probes in hydrogel closely resemble that in solution owing to the near liquid phase of hydrogel.23 However, it takes time for the analytes to reach the probes through the hydrogel network in assays without external forces like electrophoresis. Apparently, this kind of porous structure not only facilitates the entry of analytes, but also exposes more interior surfaces of the hydrogel to the analytes and hence enhances the SVR. To increase the sensitivity, detection antibodies and PEG modified SERS nanotags with RDs embedded in the gap of Au core and Ag shell were used in sandwiched immunoassay on a single PHB (Figure S1). Figure 2c and 2d show the SERS nanotag distribution in the PHB after a reaction time of 1 h, and the concentration of mouse IgG is 10 pg mL-1. The nanotags penetrate about 609 nm into the PHB, indicating that


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the top three layers of pores are utilized in our methods and the SVR of the top three layers of the PHB is calculated to be about 1.39 (Supporting Information).

Figure 2. Microscopic structure of PHB before and after immunoreactions. (a) and (b) Low and high magnification SEM images of PHB surface. (c) and (d) High magnification SEM and TEM images of PHB surface after reaction with SERS nanotags. The honeycomb grids indicate the pores of PHB with the right side as surface (d). In our method, the RDs were located in the gap between the core and shell, which acted both as labels for detection and as encoding elements for the targets. Their excitation is of vital importance for the improvement of SNR and decoding accuracy. Taking Nile blue A (NBA) dye as an example, 532, 633, and 785 nm lasers were used to excite the Raman signals of NBA encoded SERS nanotags on a PHB, respectively (Figure 3a). The best signal is obtained with 785 nm excitation. However, almost no discernible peaks are measured by 532 and 633 nm excitation. There are two main reasons, first, high fluorescence background will be gained due to the absorption of



the dye at shorter wavelength. Second, there are aggregations of nanotags in the pores of PHB (Figure 2c), resulting in the red-shift of the localized surface plasmon resonance (LSPR) and hence the excitation efficiency matched the excitation wavelength. According to our previous report, the characteristic absorption peaks of the SERS nanotags are 404 and 516 nm, which represent silver shell and gold core, respectively.15 We simulated the absorption spectra of the aggregations with different distances between nanotags (Figure 3b-e). The closer the distance, the main LSPR peak exhibits larger shift and two peaks appear. When the distance is 0.2 nm, the appeared two LSPR peaks are at 532 and 785 nm, respectively. The enhanced electric field intensity (named “hotspots”) lies the gap between the core and shell where the dyes located and the enhancement factor is about 108 at excitation wavelength of 785 nm (Figure 3d). However, for 532 nm, the “hotspots” are confined in the gaps between nanotags where there’s no dye at all (Figure 3e), contributing almost nothing to the signal. Therefore, in the following experiments, 785 nm was chosen as the excitation wavelength so that the highest SNR and sensitivity would be gained.


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Figure 3. (a) Raman spectra of NBA-SERS nanotags on reacted PHB in a sandwich immunoassay at excitation wavelengths of 532, 633, and 785 nm. The concentration of analyte mouse IgG is 10 ng mL-1. (b) Absorption cross section of the aggregated gold-silver core-shell nanoparticles (Au@Ag NP). (c) The model of three Au@Ag NPs on hydrogel substrate. Calculated magnitude of electric field at the resonant wavelength 1 (785 nm) (d) and 2 (532 nm) (e) when the gap of nanoparticles is 0.2 nm. (f) Raman spectra of different concentrations of CEA. The black dashed frames represent Raman spectra of nanotags on the PHB and NHB at the same CEA concentration of 1 pg mL-1, respectively. (g) Reference plot of intensities of Raman shift at 595 cm−1 vs. logarithm of CEA concentration. Inset is the linear part of the reference plot. Error bar is calculated from five repeats.



A quantitative measurement of CEA (0.5 fg mL-1-200 µg mL-1) was performed to evaluate the performance of the as-proposed method. Here, PHB and NBA encoded gold-silver (Au@Ag) core-shell SERS nanotags were immobilized with CEA capture and detection antibodies, respectively. The intensities of the characteristic Raman shift at 595 cm-1 of NBA were plotted against the concentrations of CEA (Figure 3f and 3g) and the linear dynamic detection range is from 1 pg mL-1 to 10 µg mL-1 (R2 = 0.955), spanning 7 orders of magnitude, which is much wider than those in literatures (Table S1).16,24 The limit of detection (LOD) and the limit of quantity (LOQ) are calculated to be 0.36 fg mL-1 and 1.33 fg mL-1 at SNR of 3:1 and 10:1, respectively (Figure 3g, inset), which are also much lower than those previously reported (Table S1).16,19,25-27 In addition, the Raman intensity of nanotags on PHB is 33.6 times of that on nonporous hydrogel bead (NHB) at the same CEA concentration of 1 pg mL-1 (Figure 3f). The number is close to the ratio of their calculated SVRs (Supporting Information). All these results prove that by the combination of PHB and SERS nanotags, ultrasensitive detection with wide LDR is realized, which enables quantitative analysis of proteins without the care of their concentration ranges and sample dilutions. Then, we immobilized AFP and CEA capture antibodies (molar ratio, 1:1) on a single PHB simultaneously and performed a multiplex immunoassay of different concentrations of AFP and CEA (1:1, v/v, from 0 fg mL-1 to 100 µg mL-1) with two kinds of encoded SERS nanotags (CV- and NBA-SERS nanotags). The mixed Raman spectra were measured (Figure 4a) and could be deconvoluted exactly with a


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multivariate deconvolution method (Figure S2) as previously reported.28 Figure 4b and 4c show the dose-responsive curves of AFP and CEA respectively. Both the responsive ranges cover more than 10 orders of magnitude and the LDRs are from 10 pg mL-1 up to 10 µg mL-1, spanning 6 orders of magnitude, which are also wider than those in previously researches (Table S1).16,19,24-27,29,30 The LODs and LOQs of AFP and CEA are then calculated to be 3.6 fg mL-1, 12.6 fg mL-1 and 1.9 fg mL-1, 6.8 fg mL-1, respectively. It could be seen that the LDR, LOD, and LOQ of the multiplex analysis on a single PHB are decreased by about one order of magnitude in comparison with that of singlet analysis. The reason is that the surface area of the PHB is occupied by multiplex probes and then the SVR is divided by the number of probe categories accordingly. Nevertheless, multiplex analysis on a single PHB is also realized with ultrahigh sensitivity and wide LDR.

Figure 4. Multiplex quantitative detection of AFP and CEA on a single PHB. (a) Raman spectra at different concentrations of AFP and CEA. (b) and (c) Dose-responsive curves of AFP (Plotted with Raman intensity at 1585 cm-1) and CEA (Plotted with Raman intensity at 595 cm-1), respectively. Inset is the linear part of the reference plot. Error bar is calculated from five repeats. In order to evaluate the selectivity of the multiplex detection on a single PHB,



another test was performed. A PHB was prepared which modified with only a single capture antibody (anti-AFP), and treated subsequently with a mixture of the two tumor biomarkers and the two kinds of SERS nanotags compounds. As shown in Figure S3a, it could be seen that one SERS signature corresponding to CV-labelled anti-AFP is observed, which indicates successful identification of the specific analyte. A similar procedure starting with anti-CEA provides signature that is predominantly from NBA-labelled anti-CEA (Figure S3b). These results demonstrate a well functional sandwich assay that provides the recognition of the SERS signatures of the correct analyte. After repeating both overall processes with five independent PHBs, a statistical measurement of reproducibility could be obtained as shown in Figure S3c, the variation coefficients of Raman signal for AFP and CEA are 7.3 % and 6.6 %, respectively. The reason is that monoclonal capture and detection antibodies are used. Another important reason is that the as-proposed bioassay is beneficial for low background, easy to wash, noninterfering feature of the components and high sensitivity from enhancement effect of SERS nanotags hybridized PHB. Finally, five different human serum samples, where the concentrations of AFP and CEA range from 1.04 to 600.8 ng mL-1 and 2.7 to 991.9 ng mL-1 as shown in Table S2, respectively, were analyzed to evaluate the practicality of the as-proposed multiplex assay for clinical application. All samples were also measured using commercial electrochemiluminescent immunoassay (ECLIA) kits in hospital. The results showed in Figure 5 demonstrate that the correlation coefficients between the two methods for multiplex detection of AFP and CEA are 99.35 % and 99.04 %, respectively, and the


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recoveries of AFP and CEA range from 91.8 % to 105.9 % and 93.8 % to 108.9 % (Table S2), respectively, indicating the accuracy of the as-proposed multiplex assay is in good agreement with that of the ECLIA. However, the LDRs of our method (both from 10 pg mL-1 up to 10 µg mL-1 for AFP and CEA) are much better than that of the ECLIA kit (LDR from 0.605 to 1210 ng mL-1 for AFP, LDR from 0.2 to 1000 ng mL-1 for CEA). What’s more, we only used 10 µL sample, which is much less than that used in ECLIA. These results strongly imply that the as-proposed assay has great potential applications in clinical analysis.

Figure 5. Multiplex analysis of clinical serum samples on a single PHB. (a) Raman spectra of real human serum samples. Correlation of detection results acquire from the as-proposed method (solid squares) and reference ECLIA method (hollow squares) for the AFP (b) and CEA (c). Error bar is calculated from five repeats. In summary, we have developed a multiplex assay on a single porous hydrogel bead with Raman dyes encoded Au@Ag core-shell SERS nanotags, which provides the basis for developing versatile protein detection protocols. The as-proposed assay addresses limitations and challenges of simultaneous multiple analytes detection. The porous structure of PHB with high surface area to volume ratio provides high probe density for target analytes, which is beneficial for the extension of concentration



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ranges to be detected. In addition, the Au@Ag core-shell structure provides stronger electromagnetic field for the signal amplification of Raman dyes. Also, the effective multivariate

deconvolution

approach

provides

accurate

and

distinguishable

identification of Raman spectra codes of multiple biomarkers in multiplex detection. The results of the multiplex detection of tumor biomarkers, AFP and CEA, indicate that the multiplex assay is robust with high sensitivity, high selectivity, and a wide linear dynamic range, as well as reliability in real clinical sample analysis. The LODs of AFP and CEA are 3.6 fg mL-1 and 1.9 fg mL-1, respectively, and their LDRs are both from 10 pg mL-1 up to 10 µg mL-1, spanning 6 orders of magnitude, which are much better than commercial ECLIA kits. All of these features open up a new window for the development of SERS-based multiplex assay in chemical analysis, healthcare, food safety, and environmental monitoring.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Experimental procedures for the fabrication of PHB carriers; preparation of SERS nanotags and TEM characterization; SERS measurement and instrumentation; sandwich immunoassay for the selection of excitation wavelength of SERS nanotags; quantitative analysis; multiplexed analysis; clinical samples analysis; Raman spectra


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with deconvolution method; electromagnetic simulations; surface area to volume ratios (SVR) calculation; selectivity evaluation (PDF)

AUTHOR INFORMATION Corresponding Authors *Email: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was financially supported by the National Key Research and Development Program of China (No. 2017YFA0205700), National Natural Science Foundation of China (Grants 21373046, 21327902, and 61675096), Program Sponsored for Scientific Innovation Research of College Graduate in Jiangsu Province (KYLX16_0284), Jiangsu Science and Technology Department (Grant No. BE2014707),

the

Natural

Science

Foundation

of

Jiangsu

Province

(No.

BK2014021828), Fundamental Research Funds for the Central Universities, Six Talent Peaks Project of Jiangsu Province, the Collaboration Research Fund of Southeast University and Nanjing Medical University (Grant No. 2242017K3DN26) and open research fund of Key Laboratory of Environmental Medicine Engineering of Ministry of Education. We are also very thankful to Chengxin Luan of Zhongda Hospital affiliated to Southeast University for real serum samples analysis.



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of

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low-abundance protein with dual nanotags. ACS Appl. Mater. Interfaces 2013, 5 (10), 4479-4485. (30) Zhang, X.; Tan, X.; Zhang, B.; Miao, W. J.; Zou, G. Z. Spectrum-based electrochemiluminescent immunoassay with ternary CdZnSe nanocrystals as labels. Anal. Chem. 2016, 88 (13), 6947-6953.


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For TOC only


Multiplex Analysis on a Single Porous Hydrogel Bead with Encoded

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