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SERS based Pump-free Microfluidic Chip for Highly Sensitive Immunoassay of Prostate-specific Antigen Biomarkers Rongke Gao, Zeyuan Lv, Yuanshuo Mao, Liandong Yu, Xiaobai Bi, Shenghao Xu, Jiewu Cui, and Yu-Cheng Wu ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.9b00039 • Publication Date (Web): 13 Mar 2019 Downloaded from http://pubs.acs.org on March 14, 2019
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SERS based Pump-free Microfluidic Chip for Highly Sensitive Immunoassay of Prostate-specific Antigen Biomarkers Rongke Gaoa,*, Zeyuan Lva, Yuanshuo Maoa, Liandong Yua, Xiaobai Bia, Shenghao Xuc, Jiewu Cuib and Yucheng Wub,* a
School of Instrument Science and Opto-electronic Engineering, Hefei University of Technology, Hefei 230009, China. b
School of Materials Science and Engineering, Hefei University of Technology, Hefei 230009, China
c
Shandong Key Laboratory of Biochemical Analysis; College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, China. KEYWORDS: Prostate cancer, Pump-free microfluidics, SERS, Immunoassay, Prostate-specific antigen ABSTRACT: Highly sensitive analysis of cancer biomarkers demonstrates an important impact in early diagnosis and therapies of cancer. A novel surface-enhanced Raman scattering (SERS) based immunoassay using microfluidic technique was reported for rapid analysis of prostate-specific antigen (PSA) biomarker. It is a useful screening test to discriminate prostate cancer and other diseases related to prostate. A “sandwich” immunoassay based on SERS nanotags, PSA biomarkers and magnetic beads was applied on pump-free microfluidic sensor. Magnetic immunocomplexes are isolated and trapped at detection chamber by a permanent magnet integrated into the chip. The PBS buffer washed magnetic immunocomplexes and brought the free gold nanoparticles to the downsteam channel for waste. Our results show a good linear response in the range from 0.01 to 100 ng mL-1. The LOD of PSA level is estimated to be below 0.01 ng mL-1 using this chip. This detection levels of PSA biomarker in human serum can be accomplished in 5 min without manually incubation and heavy syringe pump. To the best of our knowledge, this is the first SERS-based immunoassay which applied pump-free microfluidic chip as detection platform. We believe that the proposed method reveals a valuable potential tool for the diagnosis of prostate cancer.
Prostate cancer (PCa) has been one of the most general causes of cancer death for the men.1 The prostate-specific antigen (PSA) is a kind of kallikrein-like serine protease, and is approved by the US Food and Drug Administration (FDA) associated with prostate cancer diagnosis and prognosis as biomarker.2 The PSA levels are increased in the serum of patients, when prostatitis, benign prostate hyperplasia (BPH), and PCa were happened in the prostates. Especially, PSA levels of old men is also increased to abnormal level as increased age. Identification of PCa, BPH and normal sample has been attracted to many researchers.3 To provide the screening possibility of prostate cancer before prostate biopsy, the amount of PSA is used as reference to medical doctor in general prostate cancer diagnosis. The PSA in serum above 10.0 ng mL-1 manifests serious potential of PCa. In contrast, it shows low potential of PCa when the PSA is below 4.0 ng mL-1.4 In this case, a rapid and portable detection method of PSA level is urgently desiderated to precisely screen PCa for patients as early diagnose.
photo-bleaching effects, time consuming and laborious remain drawbacks for real-time performance of point-ofcare testing (POCT) application. Surface-enhanced Raman scattering (SERS) detection have become a promising and powerful spectroscopy technology by providing ultrasensitive and intrinsic chemical fingerprint information.10-13 Therefore, SERS detection technology for rapid and sensitive disease diagnostic tends to incrementally popular. Several works have demonstrated that SERS-based immunoassay technique for PSA detection can improve the diagnosis accuracy in PCa.14-15 Some problems of other general detection techniques were solved by this SERS-based immunoassay. Nevertheless, the SERS-based detection still has to suffer from interminable manual incubation and washing time in benchtop experiments. Additionally, the magnetic immunocomplexes should be manually isolated by magnetic bar leading to inconsistent assay condition. To achieve rapid immunoassay and reproducible SERS signals, microfluidic chip as a new detection platform was applied in SERS-based assays.16-18 Uniform mixing and continuous assay steps were sufficiently executed on a centimeter-size chip. Recently, our research group reported a SERS-based microfluidic chip for sensitive immunoassay of PSA biomarker in automatic manner,
To date, various detection methods, such as fluorescence immunoassay,5 photoelectrochemical 6 immunosensor, electrochemiluminescence,7 mass spectrometry8 and amperometric immunoassay9 have been used for the direct detection of PSA. However, the
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which only cost 5 min without washing step.19-20 Microfluidic technique has shown small reagent consumption, fast mass transfer, automation and portability benefitting from microscale channel and high surface to volume ratio.21-23 The combination of SERS with microfluidic techniques has indicated several helpful advantages, such as reduced detection time and automatic sampling, over general measurement condition. These advantages significantly contributed to quantitative SERS analysis and received attractive focus from many research communities. Therefore, SERS-based microfluidic platform was broadly employed for biological samples as a biosensor.10, 24-25 The conventional microfluidic system required external pumps, connectors, and control facility. It significantly increased cost and limited the application in field and home test. To further miniaturize the system, pump-free microfluidic technology were developed, such as degas, capillary and finger pumps.26-27 Dimov et al. demonstrated a degas-driven microfluidic analysis system to perform biotin–streptavidin test using raw whole-blood sample.28 Shin et al. integrated capillary and pressure pump with microfluidic device to achieve colorimetric bioassays of glucose and albumin. They offered an obvious example to show pump-free device as a powerful tool in practical application.29 A user-friendly finger-actuated microfluidic device was investigated for the blood cross-matching test by precisely controlling the pressure in channel.30 However, these pump-free approaches underwent low sensitivity, insufficient flow control and complex fabrication progress. It has a demand of a simple, robust and low cost pump-free microfluidic chip for sensitive SERS detection.
Fig. 1. Schematic illustration of the pump-free microfluidic chip for the detection of PSA biomarkers. The chip contained three functional compartments: (i) samples mixing and immunoreaction, (ii) storage and detection, (iii) capillary pump. The photograph showed all microfluidic channel was filled with red and blue inks.
Herein, we proposed a novel pump-free microfluidic sensor for SERS-based immunoassay of PSA biomarker. The aqueous phase including PSA biomarkers and magnetic beads (MBs) were automatically flowed, mixed and reacted the entire channel by a capillary pump on chip. It greatly reduces the sensor size and improves the portability, because the heavy syringe pump related
equipment in conventional microfluidic system was replaced. The level of PSA biomarker was measured by SERS at detection chamber where the immunocomplexes was isolated by a permanent magnet. To the best of our knowledge, we firstly applied pump-free microfluidic chip to SERS-based immunoassay as detection platform. The SERS based pump-free microfluidic sensor realized highly sensitive detection of PSA automatically and rapidly.
Experimental section Chip design and fabrication A pump-free PDMS microfluidic chip was developed to integrate the permanent mini-magnet into an immunoassay process and SERS detection. The resulting SERS based pump-free microfluidic chip (Fig. 1) consists of three components: i) A 3 mm round chamber was used for pre-mix solutions and winding-shaped structure was incorporated into the channel for further mixing and reaction zone. The stretching and folding of the fluids induce a chaotic advection effect, which increases species mixing. This part was used to promote the formation of immunocomplexes by the reaction of antibodies and PSA antigen. And ii) a 1.6 mm length rectangular chamber along the right side channel of T-junction was used for storage and detection of immunocomplexes. A 4.0 mm round mini-magnet was placed beside the chamber. The third compartment, a comb-like structure channel, acts as a capillary pump to drive fluids through the entire channel. Meanwhile, the hydrophilic channels led the movement of fluid upon contact such that 80 μL sample could be loaded about 60s. The chip has three inlets for sample and PBS wash buffer. One outlet is located directly after capillary pump, through which fluid waste passes. The fabrication of microfluidic chip utilized the rapid prototyping and UV photolithography methods. A negative SU-8 3035 photoresist mold was fabricated using a photomask on a clean silicon wafer. It was followed by developed in SU-8 3035 developer (Microchem Corp, Westborough, US) and post-baked. The silicon wafer was washed by acetone and deionized water to remove residual photoresist, and then dried with nitrogen gas. Whereafter, the PDMS prepolymer and curing agent were mixed in a 10:1 ratio by weight and then removed the air bubbles using vacuum pump. The PDMS polymer was cast into the mold using a petri dish. To perform the capture process of magnetic beads, a N45 permanent magnet was inset alongside the detection chamber before heating. A 4.05 mm round fence on the mold can fix the location of magnet. It was heated at 70 ◦C in oven for 2 h, and the PDMS replica including magnet then peeled off from the mold. Inlet and outlet holes (D=4mm) for fluid injection were punched in the PDMS layer. Hydrophilic surface treatment was performed by coating PEG polymer on the PDMS layer.31 Briefly, PDMS replica and glass slide were respectively cleaned using an ultrasonic wash in isopropanol and acetone for 5 minutes, rinsed by DI water, and dried on heating plate. PDMS replica was exposed to an oxygen plasma for 90 s and immediately modified by the PEG, then moved to the hot plate at 150 ◦C for 25
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ACS Sensors minutes. Following heating, the PDMS replica was washed by isopropanol and DI water respectively at room temperature to remove the residual PEG, and dried on the hot plate at 70 ◦C. Then, the PDMS replica was attached onto a glass slide. The width and depth of microfluidic channel were 200 µm and 100 µm respectively. To ensure that loading effects did not affect the results, new chip was utilized for each measurement. Microscope images and Raman measurements Bright field images of immunocomplexes capture process and fluorescence images of mixing efficiency test were obtained by Eclipse Ti-U inverted research microscope with DS-Qi2 monochrome camera (Nikon, Japan). The eppendorf pipette was used to inject samples into the chip. Images profile of mixing efficiency test in microfluidic chip were recorded and analysed by Nikon NIS-Elements imaging software. Raman spectra were taken by LabRam HR Evolution system (HORIBA Jobin Yvon, France) with a 10 mW helium/neon laser operating at λ=632.8 nm. The Rayleigh line was eliminated from the excited Raman signal by a holographic notch filter located in the collection path. The resolution of Raman spectral was 1 cm-1. The laser spot was focused on the magnetic immunocomplexes by a 10× objective lens (numerical aperture=0.25) in storage and detection rectangular chamber. The accumulation time was 10 s and the laser spot size was 3 µm. LabSpec 6.0 software was used for Raman system manipulation and data acquisition. Baseline correction has been performed to suppress background noises. Fig. S1 showed the SERS measurement setup for the pump-free PDMS microfluidic chip.
PBS buffer and flowed through capillary pump to outlet. The laser beam was focused on accumulational immunocomplexes, and SERS signal were collected for sensitive detection of PSA biomarker. A photograph of the pump-free PDMS microfluidic chip (40 × 21 mm) flooded with blue and red food dye solutions was displayed in Fig. 1. The microfluidic channel was rendered hydrophilic character by the PEG coating. As demonstrated in Fig. 2, red and blue inks automatically flowed in the microfluidic channel to test the robustness and hydrophilic property, and no leakage was found during experiments. In our experiments, long-term stability of hydrophilic microfluidic channel surface maintained in 30 days at room temperature, and it did not exert obvious influence on the hydrophilic property. Consequently, the approach mentioned above are able to achieve liquid autonomous transportation in the channel by the capillary force without using external pumps. A 150 μl inks can fill out the channel within 55s, as shown in Fig. 2.
Results and discussion Characterization and workflow of pump-free PDMS microfluidic chip Highly sensitive detection of PSA biomarker is essential for early diagnosis of prostate cancer. A couple of papers reported the SERS-based immunoassay for PSA detection, but it is still onerous in performance process resulting from several problems containing manually preparing steps for assay, inconsistent assay conditions and heavy syringe pump. To resolve these problems, a new designed pump-free PDMS microfluidic chip realized SERS-based immunoassays of PSA biomarker in an automatic manner. The workflow was introduced in Fig. 1. MBs, PSA biomarkers and SERS nanotags were added into the channel through three inlets to identify the levels of PSA. Once all aqueous solutions contact with the channel surface, it was driven by capillary force and flowed along the channel. Followed by pre-mixing in round chamber, the aqueous solutions completed the mixing process due to the stretching and folding of the fluids in windingshaped structure. MBs, PSA biomarkers and SERS nanotags efficiently generated immunocomplexes as sandwich type during this step. Then, the immunocomplexes were attracted to the right side channel and captured in detection chamber since the mini-magnet was located there, as shown in Fig. 5a. The unbonded SERS nanotags and PSA antigen was washed by
Fig. 2. The photographs of red and blue inks automatically flowed by capillary force in microfluidic channel according to time.
Fig. 3. Fluorescence microscope images of the entire microfluidic channel. The yellow dotted arrows indicated the profile locations using NIS-Elements software and four corresponding fluorescence intensity profiles were shown at both sides. DI Water flowed in the middle part of the channel (black color), and FSS solution flowed in the two side parts (green color). X and Y axes represent the distance and the fluorescence intensity.
Validation of mixing efficiency of the microfluidic chip
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Rapid and efficient mix intensely effect the quantitative SERS analysis result as we described before. To validate the mixing performance of this chip, the variations of fluorescence intensity cross the fluid were taken at the six positions along the microfluidic channel. Fig. 3 demonstrates the fluorescence image of whole chip recording by Eclipse Ti-U inverted research microscope. The 3 × 10-4 M FSS and DI water were excited by blue laser and showed green fluorescence to evaluate mixing efficiency. At the position #i and #ii, the aqueous stream was divided by three parts clearly due to laminar flow regime and the corresponding fluorescence profiles showed low intensity at the center of channel in Fig. 3. From position #iii, it was found that a number of FSS molecules slowly appear in the DI water stream because the molecule diffusion is the main force in laminar flow. However, the winding channel was embedded to introduce the chaotic advection of fluids. It accelerated the FSS molecules movement in channel and the aqueous stream completely mixed at winding channel area (# vi). The signal-to-noise ratio was 371.63. The SERS signal were measured at different locations along the microfluidic channel as shown in Fig. S2a. According to peak intensity at 1614 cm−1, it exhibited that the mixing process of SERS nanotags and DI water was almost complete by the point # v (Fig. S2b). Therefore, the fluids sufficiently mixed within the winding channel area. SERS-based microfluidic immunoassay To validate the immunoassay process on microfluidic chip, two channel positions were selected for SERS measurements, which represented before and after the immunoreaction, as shown in Fig. 4a. Herein, red laser spot was focused on the inlet channel of capture antibodies conjugated magnetic beads (i) and immunocomplexes in detection chamber (ii), and their corresponding SERS spectra were displayed in Fig. 4b. SERS signal of MGITC was only detected on position (ii) when the MGITC labelled AuNPs were connected to magnetic beads as immunocomplexes. In the other cases of capture antibodies conjugated magnetic beads, the intrinsic Raman peaks of the PDMS (485 cm-1, 612 cm-1, and 705 cm-1) were also found in the spectra, and the signal intensity were negligible compared with immunocomplexes. To quantitatively evaluate the PSA level, the 1614 cm-1 peak was selected as main reference, which is located in different area with the intrinsic peaks of PDMS. Consequently, it could be concluded that this chip was available for trace analysis of PSA. Fig. 5a shows the process of magnetic imunocomplexes captured and stacked in the detection chamber by a series of microscope images. The mini-magnet was applied to draw the imunocomplexes by its intrinsic magnetic field, and the image was recorded when the imunocomplexes reached to chamber. It takes the advantage of permanent magnet which has strong magnetic field and easily embedded within microfluidic chip. Furthermore, the stacked imunocomplexes greatly increased the detection sensitivity since the immobilization amount of the capture antibodies is observably aggrandized on threedimensional magnetic bead over one-dimensional plate.
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It can contribute more Raman signal of SERS nanotags in one measurement to improve the sensitivity. Movies S1 demonstrated the real-time isolation of the magnetic immunocomplexes and the stack process. The SERS signal was measured every minute since the magnetic immunocomplexes initially accumulate to determine the enough stack time. Fig. 5b demonstrated that the amount of immunocomplexes was sufficient for SERS detection at the third minute due to the intensity of 1614 cm-1 peak saturated.
Fig. 4. (a) The picture of pump-free microfluidic chip and two SERS detection points. (b) The corresponding SERS spectra for (i) capture antibodies conjugated magnetic beads and (ii) immunocomplexes contained SERS tags.
Fig. 5. (a) Microscopic images of the immunocomplexes capture process in the detection chamber as the function of time, and (b) their corresponding SERS intensities of 1614 cm-1 peak. The standard deviations are from five SERS measurements.
Quantitative analysis of PSA using the pump-free PDMS microfluidic chip To evaluate the detection performance of our chip, quantitative analysis of the PSA biomarker was applied. Various concentrations of PSA were introduced to the chip, and formed the imunocomplexes with magnetic beads and SERS nanotags in microfluidic channel. The laser spot was fixed and focused on detection chamber of each chip to collect SERS spectra in 10s for measurement. SERS spectra of the stacked imunocomplexes at the detection chamber were detected and characterized for validation, as shown in Fig. 6a. It should be noted that SERS nanotags and magnetic beads intended to deposit on the internal channel for a long detection period, which known as loading effects. This effect can affect quantitative analysis results. To avoid loading effects, new chip was used for each measurement. The featured SERS peak of MGITC (1614 cm-1) was selected for monitoring PSA levels. The SERS intensity as the function of concentration is plotted in Fig. 6b. PSA concentration from 0.01 to 100 ng mL-1 portrayed a good linear response
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ACS Sensors (R2=0.986) with the error bar from five measurements. The LOD was as low as 0.01 ng mL-1. Importantly, this detection level meet the requirements demanded in clinical applications. Compared with conventional ELISA method, our result demonstrated significant capability of highly sensitive detection on a microfluidic chip.32 This PSA sensor can analyse the PSA level below the clinically threshold value of 4 ng mL−1 and indicate the risk of PCa. Therefore, the proposed SERS-based immunoassay technique using pump-free PDMS microfluidic chip has attractive potential for a fast and sensitive screening test of prostate cancer.
Supporting Information Available: The following files are available free of charge. Experimental details. Preparation procedure of SERS tag and antibody conjugation, schematic of experimental setup comprising the pump-free microfluidic device and Raman instrument, optical arrangement for focusing the laser on the detection chamber of the channel. Mixing efficiency test of microfluidic chip using SERS. Movie S1. Capture and stack process of magnetic immunocomplexes.
AUTHOR INFORMATION Corresponding Author Rongke Gao, E-mail addresses:
[email protected] Yucheng Wu, E-mail addresses:
[email protected] Author Contributions All authors have given approval to the final version of the manuscript.
Notes The authors declare no competing financial interest.
ACKNOWLEDGMENT Fig. 6. (a) The SERS spectra on the basis of PSA concentrations on pump-free microfluidic chip and (b) corresponding calibration curves. The concentration ranges of PSA was 0.01-100 ng mL-1. Variations of SERS intensities of 1614 cm-1 peak were utilzed for PSA quantitative analysis. The standard deviations are from five SERS measurements.
Conclusions We developed a novel SERS-based microfluidic immunoassay chip for highly sensitive analysis of PSA biomarker. For sensitive detection and reducing the sample consumption, a pump-free PDMS microfluidic chip was proposed and fabricated to execute the PSA immunoanalysis with SERS in a integrated automatic manner. The pump-free PDMS microfluidic chip contained three parts: rapid mixing and immunoreaction, SERS detection and capillary pump. The technique demonstrate a “sandwich” immunoassay of a cancer biomarker on chip without external pump, because the aqueous fluids can be driven by capillary pump with comb-like structure channel. In this microfluidic chip, the immunocomplexes were efficiently captured from microfluid using a mini-magnet placed beside the detection chamber. The SERS signals of imunocomplexes were detected and performed for the quantitative analysis of PSA levels using the proposed pump-free PDMS microfluidic chip. The LOD determined by this highly sensitive detection platform was as low as 0.01 ng mL−1. This LOD level demonstrates that this SERS-based pump-free PDMS microfluidic immunoanalysis is highly qualified for the clinical diagnosis of PCa. Therefore, the presented SERSbased pump-free PDMS microfluidic immunoanalysis indicated high sensitivity and accuracy, which is a promising early screening test for prostate cancer.
ASSOCIATED CONTENT
This work is supported by the National Natural Science Foundation of China (No. 61601165). We also acknowledge financial support from the China Postdoctoral Science Foundation (No. 2018T110613), the Fundamental Research Funds for the Central Universities (No. JZ2016HGBH1052), the Anhui Key Project of Research and Development Plan (No. 1704d0802188). This work was also partially supported by the Open Project of Faculty of Chemistry of Qingdao University of Science and Technology (QUSTHX201805).
ABBREVIATIONS SERS, Surface-enhanced Raman scattering; POCT, Point-ofcare testing; FDA, Food and Drug Administration; BPH, Benign prostate hyperplasia; PDMS, Polydimethylsiloxane; PCa, Prostate cancer; PSA, prostate-specific antigen; MBs, magnetic beads; LOD, limit of detection; MGITC, malachite green isothiocyanate; PEG, poly (ethylene glycol); FSS, fluorescein sodium salt; AuNPs, gold nanoparticles; ELISA, enzymelinked immuno sorbent assay.
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