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Design and preparation of centrifugal microfluidic chip integrated with SERS detection for rapid diagnostics
Author’s Accepted Manuscript Design and preparation of centrifugal microfluidic chip integrated with SERS detection for rapid diagnostics Xi Su, Yi Xu, Huazhou Zhao, Shunbo Li, Li Chen www.elsevier.com/locate/talanta
PII: DOI: Reference:
S0039-9140(18)31164-0 https://doi.org/10.1016/j.talanta.2018.11.014 TAL19247
To appear in: Talanta Received date: 28 July 2018 Revised date: 26 October 2018 Accepted date: 5 November 2018 Cite this article as: Xi Su, Yi Xu, Huazhou Zhao, Shunbo Li and Li Chen, Design and preparation of centrifugal microfluidic chip integrated with SERS detection for rapid diagnostics, Talanta, https://doi.org/10.1016/j.talanta.2018.11.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Design and preparation of centrifugal microfluidic chip integrated with SERS detection for rapid diagnostics Xi Sub,c, Yi Xua,b,c,*, Huazhou Zhaob,c, Shunbo Lia,c, Li Chena,c,* a
Key Disciplines Laboratory of Novel Micro-Nano Devices and System
Technology, Key Laboratory of Optoelectronic Technology and Systems, Ministry of Education, School of Optoelectronics Engineering, Chongqing University, Chongqing 400044, China b
School of Chemistry and Chemical Engineering, Chongqing University,
Chongqing 400044, China c
International R & D center of Micro-nano Systems and New Materials
Technology, Chongqing 400044, China [email protected] [email protected]
*Corresponding author: Abstract A microfluidic SERS chip integrated with blood separation and in-situ detection was designed and fabricated for the rapid detection of clinical blood samples. Each functional unit in the microfluidic SERS chip consist of separation-decantation cavity based on centrifugal separation principle, mixing channels and SERS detection chamber built with integrated nano-Au on Ag film microstructure. The serum creatinine was selected as a typical sample to demonstrate the capability of microfluidic SERS chip. It was found that the creatinine SERS characteristic peaks at 678 cm-1 can be effectively identified and the detection limit could be as low as 4.42 × 10-3 μmol mL-1 in water. The blood samples were also tested in microfluidic SERS chip. The whole separation and test process could be completed within 2 min, which is a significant improvement in the field of creatinine detection. The whole blood of six cases clinical blood samples were also tested, and the results were consistent with the enzymatic results. The developed microfluidic SERS chip has advantages including reduction of the required quantity of blood sample, reusable and easy to operate. It is expected to provide a new method for rapid diagnostics.
1
Graphical Abstract
Key words: Surface enhanced Raman spectroscopy (SERS), Ag film@nano-Au substrate, centrifugal microfluidic chip, blood separation, rapid diagnostics
1. Introduction The micro-Total analysis system (µTAS) developed very rapidly in the field of point of care in last decades due to its unique advantages such as less sample volume, fast detection, high throughput, low contamination and easy integration ect. Lots of analytical methods including electrochemistry[1], UV-vis[2], fluorescence[3] and Raman/SERS[4-6] have been applied in the whole blood test, after the pretreament of serum extraction. Compared to conventional techniques, microfluidic analytical method is more popular in clinical blood test due to the high efficiency and elimination of large scale apartus, such as LC、HPLC、LC-MS、GC-MS and so on. The serum extraction is a crucial step in whole blood test since the blood cells could interfere with the detection of the target molecules. The common methods for the separation of blood in microfluidic chip include physical method based on particle size[7], electrophoresis[8] and centrifugation separation[9-12]. The separation based on particle size has the problem of channel blockage by blood cells, as well as the low separation efficiency [7, 13]. Electrophoretic separation technology has good analytical efficiency, however, it has inevitable cell damage and hemolysis phenomenon caused by the high driving voltage, and it is difficult to obtain the 2
original and stable serum samples[8]. Existing studies have shown that the centrifugal microfluidic technology can obtain high quality separation serum, based on the separation principle that the mass density of each blood component is different[14]. Chen et al[15] designed an integrated chip system with separation-decantation microchannel network to separate serum from the whole blood. However, only the diluted blood sample could be separated to obtain high quality serum in this chip, and the obtained serum used for detection was less than 1 μL, which was below the required sample amount. Amasia et al[16] presented a new device with larger centrifugal disc for serum separation within about 5 min, which could deal with 2 mL of undiluted blood samples at a time and obtain high quality serum. The potential and advantages of centrifugal microfluidic technology in blood separation were showed by above researches. However, a mature microfluidic chip integrated with blood separation and detection units needs to be studied and expanded. Surface Enhanced Raman spectrum (SERS) can greatly enhance Raman signal due to the strong interactions between moleculars and the surface of rough metal nanostructures[17]. SERS, with features of high sensitivity, non-interference by water and transparent packaging material, has a distinct advantage on fast identification and analysis of complex blood samples [17-19]. Gold and silver nanoparticles were the most widely used metal among SERS materials. The Ag nanostructure has a strong SERS effect[20]. However, the Ag NPs were very active, and the surface could be easily oxidized, while the Au NPs have good stability and biocompatibility. The oxidation of the Ag NPs film can be prevented, if a layer of Au NPs were assembled on the surface of the silver NPs film, and the interaction between the substrate and biological samples can be enhanced[21, 22]. Therefore, microfluidic chip technology and SERS technology were combined to form integrated microfluidic SERS chip to realize high efficient separation and analysis of blood, which is the new trend and research hotspot of developing the clinical rapid detection field[23]. According to the demand of rapid diagnostics, an integrated microfluidic SERS chip including blood separation and SERS in situ detection was designed and prepared. The centrifugal microfluidic technology and SERS technology were well organized
in
the
developed
microfluidic
SERS
chip,
which
consist
of
separating-decantation cavity, mixing channel and detection cavity. Nano-Au on Ag film SERS substrate was designed and prepared in the detection cavity to improve the sensitivity. The serum creatinine was selected as the test object. The functions of 3
separation and detection in microfluidic SERS chip were selected and optimized, and a new method for serum test was established. The reliability of the developed device was proved using blood samples from healthy person and patients with chronic renal failure. A new method for the diagnosis of clinical nephropathy was provided by the established testing method used microfluidic SERS chip.
2. Materials and methods 2.1 Materials Hydrogen tetrachloroauric acid (HAuCl4·4H2O, AR) was purchased from Sinopharm chemical reagent Co. Ltd (Shang Hai, China). Silver nitrate (AgNO3, AR) was purchased from Aladdin (Shang Hai, China). Stannous chloride (SnCl2, AR), Trisodium citrate (AR), glucose (AR), Ammonia water (NH3·H2O, AR), Hydrogen peroxide (H2O2, 30%), Concentrated sulfuric acid(H2SO4, 98%),
Concentrated
hydrochloric acid (HCl, AR), Concentrated nitric acid(HNO3, AR) were purchased from Chong Qing Chuandong Chemical (Group) CO., LTD. The SU-8 photoresist and developer was purchased from Microchem, and poly(dimethylsiloxane) (PDMS) from Dow Corning (Sylgard 184). Blood from healthy people was obtained from Hospital of Chongqing University, blood from patients with chronic renal failure was provided by Southwestern Hospital. 2.2 Sample preparation Creatinine samples with concentrations of 8.84 × 103 μmol mL-1, 1.18 × 104 μmol mL-1 and 1.33 × 104 μmol mL-1 were prepared by dissolving 0.1000g、0.1332g、 0.1500g standard creatinine powder in deionized water respectively. Then, the solution with 8.84 × 103 μmol mL-1 creatinine was diluted into a series concentration of creatinine solution from 4.42 × 10-3 to 44.2 μmol mL-1. The blood from healthy people with creatinine were prepared by mixing the blood with the as-prepared creatinine solutions (1.6 × 103, 3.2 × 103, 4.8 × 103, 8.0 × 103, 1.12 × 104, 1.28 × 104 μmol mL-1) with 1:1 ratio to obtain the desired concentrations. 2.3 Instrumentations Confocal Raman (HORIBA LABRAM, France) equipped with a 633 nm He–Ne laser as excitation source was used for detection. The power of the excitation source was about 17 mW, and the spectral resolution of this instrument was 2 cm-1. The 600 gr•mm-1 gratings and a neutral density filter of 10% were used during the detection. The Raman spectra were collected through a 50× microscope objective lens (NA 0.5) 4
and acquisition time of 4 s was used. The blood separation was realized by SJT-1 Spinner purchased from Shanghai Xueze Optical Machinery Co., Ltd. The surface morphology of Nano-Au on Ag film substrate were characterized using scanning electron microscopy (SEM, Hitachi 7800F system, Japan). The samples were pumped into the chip by Flow injection pump (HARWARD，USA). 2.4 Preparation of Microfluidic SERS chip The Microfluidic SERS chip consists of two parts: glass substrate and PDMS cover.
Glass
substrate
with
microchannels
was
prepared
using
standard
photolithography and wet etching technology. The cover was prepared by mixing PDMS prepolymer and curing agent, pouring onto a cleaned and smooth glass plate, and cured for 2 h at 90 ℃. Then, the inlets and outlets of chip were punched for tubing connection. The prepared glass substrate was merged in hot piranha solution for 30 min to make the surface hydroxylation, then rinsed by deionized water and ethanol. The PDMS cover was cleaned by means of ultrasonic cleaning for 5 min in the deionized water and ethanol, and dried by nitrogen. Glass substrate and PDMS cover were assembled, and placed under 75 ℃ for 10 min to strengthen the bonding force between them. Finally, bonding based on physical function between the substrate and cover plate was realized. The nano-Au on Ag film SERS substrate was prepared by self-assembly method.[24] Firstly, SnCl2 solution (0.02 wt%) was introduced in the SERS detection chamber to activate the glass substrate. The newly prepared silver-ammonia solution and 5% glucose solution were mixed by the volume ratio of 1:5, then the mixture was pumped to the SERS detection chamber in a 60 ℃ water bath for 10 min to form shining silver mirror. In order to obtain self-assembled AuNPs on the Ag film, the SERS detection chamber was immersed in 1% cationic polymer PDDA for 30 min, rinsed by DI water, and the gold sol (Hydrogen tetrachloroauric acid) was injected to react for 6 h. It was noted that gold sol was renewed every 30 min. The microfluidic SERS chip integrated with nano-Au on Ag film SERS was prepared. 2.5 Microfluidic SERS chip test 3 μL blood was pumped into each separation chamber of microfluidic SERS chip for blood separation test. The chip size parameter, rotation speed and time were optimized after observing clear serum separation in the separation-decantation cavity 5
in the device. The standard creatinine powder and 8.84 × 103 μmol mL-1 creatinine solution were tested by the microfluidic SERS chip to determine characteristic peak of creatinine molecular, deionized water and serum from healthy people were used as background signal. Different concentrations of creatinine solution or healthy blood contained different concentrations of creatinine were tested in microfluidic SERS chip. The detection effect was evaluated by averaging spectra from three times test of each sample. The serum SERS spectra were used to establish the relationship between the creatinine concentration and characteristic peak intensity. In addition, six clinical blood samples were tested by the microfluidic SERS chip method respectively. The conventional enzyme method was also performed for comparison between two methods.
3. Results and discussion 3.1 The microfluidic SERS chip The designed and fabricated microfluidic SERS chip with double function of separation and SERS in situ detection consists of three parts: separation-decantation cavity, mixing channel and SERS detection chamber (Fig. 1). There were four pairs (8 units) of channels symmetrically distributed on the device. The size of the structure was shown in Fig. 1, and the depths were 80 μm for all the channels. When microfluidic SERS chip was rotating, blood sample in the separation cavity was affected by three kinds of force - centrifugal force (Fw) produced by the system rotation, centripetal force (Fc) by the systems and fluid velocity and centrifugal force (Fr) by the curvature of the micro-channels[25]. The different densities between the blood cells and serum brought out the different net forces. Cells with larger density experienced larger net force and deposited on the bottom of the separation chamber. On the contrary, serum with smaller density was located in the upper part. By controlling the centrifugal rate, the separated serum was easily imported into the decantation cavity through the micro-channel between the separation cavity and decantation cavity, so that the blood cells and serum were separated [26, 27]. The hole on the decantation cavity could balance the pressure of atmosphere. The serpentine mixing micro-channels extended to the direction of the center, which connected the decantation cavity and SERS detection chamber. The separated 6
serum could be imported into the SERS detection chamber by negative pressure through the external pipe of microfluidic SERS chip’s center. The mixing channels were beneficial to realize a variety of samples mixing quickly and sufficiently under the condition of micro-scale. In particular, the mixing efficiency could be optimized by adjusting the length of the channel[28]. In order to achieve fully mix, the length of the serpentine channel was set to about 36,840 μm and the channel width was 200 μm. Negative pressure was applied at the end of the detection chamber to direct the serum to the decantation cavity, and then flow into the detection chamber integrated with nano-Au on Ag film substrate for SERS detection. Owing to the design of separation chamber, there is no interference of Raman signal caused by the blood cells. The sensitivity of SERS detection signal could be improved effectively through the synergistic effect among Au nanoparticles and between Au nanoparticles and Ag film.[21] The key and difficulty in microfluidic SERS chip was the integration of nano-Au on Ag film on the glass, so self-assembly method, which was easy to carry out and realize integration in microchannels, was introduced. A layer of Ag nano film was deposited on the surface of the detection chamber by self-assembly method described in part 2.3, the SEM image of Ag film showed that islands of silver particles were formed after 10 min of the silver mirror reaction (Fig. 2a). Then Au nanoparticles were assembled on the Ag film, and the Au nanoparticles（~100 nm） was uniformly distributed on the surface of Ag islands (Fig. 2b), and finally the nano-Au on Ag film SERS substrate was integrated in the SERS detection chamber successfully. 3.2 Optimization of the separation process Due to the total volume of the separation cavity was 2.24 μL by calculating. 3 μL of blood was selected as the optimized amount for processing. If the amount of blood sample was excessive and beyond the capacity below the liquid outlet, blood cells might flow into the mixing channels, serum and blood cells could not be separated well. On the contrary, the serum and blood cells would be remained in the separation cavity and the serum could not be separated into the decantation cavity. The initial width (X in Fig. 1) of the micro-channel connecting the separation and decantation cavity is an important parameter which determines whether the serum above the separation chamber could flow smoothly into the decantation cavity. In order to optimize the separation of microfluidic SERS chip, the vertical distance X was optimized. So as to select the most appropriate width, three kinds of width were 7
designed. X was 1 mm in 1 and 5 unit, 2 mm in 2, 4, 6 and 8 unit, and 3 mm in 3 and 7 structure unit. The centrifugal speed was set to 1000 rpm for 100 s to ensure that the blood cells were not ruptured after loading blood samples. The separation results were showed in Fig. 3. The blood in unit 1 and 5 were stayed in the separation cavity (Fig. 3a), and with the increase of the spin time, serum - blood cells were distributed in two layers in the separation cavity, but the serum could not enter the decantation cavity. The serum - blood cells in unit 2 and 6 were separated well (Fig. 3b), and serum with a spot of blood cells flowed into the decantation cavity. The small amount of blood cells will stay on the bottom of the decantation cavity, so there was no interference to the test. In unit 3 and 7, a large number of blood cells entered into the decantation cavity (Fig. 3 c), and the separation effect was poor. It was not possible for the subsequent serum test. Therefore, 2 mm was optimum for the initial vertical distance (X) of the channel between the separation cavity and the decantation cavity. 3 μL blood was tested in microfluidic SERS chip for spin time of 20, 40, 60, 80, 100 s to optimize the separation time (Fig. 4 a-e). As the separation time was increased, there were more and more blood flowed into the separation cavity from the input port. When the separation time was 80 s, the blood was nearly full in the separation cavity. And when 100 s was applied, all of the 3 μL blood filled into the separation cavity, the blood serum and blood cells were separated perfectly, and the vast majority of serum and a small number of blood cells flowed into the decantation cavity for the subsequent SERS test of serum. Therefore, the optimized centrifugal separation time was identified to be 100 s. 3.3 Performance of the SERS detection on the microfluidic chip The creatinine standard powder was tested with Raman to confirm the characteristic peak of creatinine detected by microfluidic SERS chip, and the results were shown in Fig. 5. The characteristic peak at 678 cm -1 comes from shear vibration of the ring plane, stretching vibration of C - NH2, the ring and C - O. It was used for the trademark peak of creatinine [29]. Deionized water and creatinine aqueous solution with concentration of 8.84 x 103 μmol mL-1 were tested in microfluidic SERS chip integrated with nano-Au on Ag film substrate, and the SERS signals were collected (Fig. 6). The background peaks of the substrate were mainly concentrated at 784 cm -1, 715 cm -1, 1001 cm -1, 1395 cm -1, 1240 cm -1, 1544 cm -1, and 1456 cm -1, and most of the peaks were weak, so the characteristic peak of creatinine would not be interfered by the background peaks. The above results illustrated that the microfluidic 8
SERS chip could be used for creatinine SERS test. The detection sensitivity in microfluidic SERS chip for creatinine molecule was also evaluated. creatinine solution with concentration of 4.42 × 10-3, 4.42 × 10-2, 0.442, 4.42 and 44.2 μmol mL-1 were tested in microfluidic SERS chip respectively, the results were shown in Fig. 7. With the reduction of the creatinine concentration, the SERS intensity at the characteristic peak of creatinine (678 cm -1) was gradually reducing. The characteristic peak could still be identified when the concentration of creatinine was as low as 4.42 × 10-3 μmol mL-1, suggesting that the detection limit of creatinine using the as-prepared nano-Au on Ag film SERS substrate was 4.42 × 10-3 μmol mL-1. 3.4 Serum creatinine detection by microfluidic SERS chip In order to verify that microfluidic SERS chip could be used for serum creatinine detection, the healthy human blood and the healthy human blood with different concentrations of creatinine were tested. The separated serums were pumped into the detection chamber for SERS test. The SERS sepctra of the separated creatinine were collected as shown in Fig. 8. It was illustrated that the characteristic peak of creatinine at 678 cm-1 was observed, and the peak intensity was increased with the increase of creatinine concentration, but there was no obvious linear relationship between peak intesity and creatinine concentration. The detection limit of creatinine using the as-prepared nano-Au on Ag film SERS substrate was 8.0 × 102 μmol mL-1. It was noted that the peak strength of the characteristic peak of creatinine was affected by the background peak of nano-Au on Ag film substrate and the complex serum composition, resulting in a higher detection limit for serum creatinine detected by this method[30]. The precise quantitative analysis of serum creatinine by this microfluidic SERS chip was difficult in the certain degree, but the chip could be used for auxiliary diagnosis of chronic renal failure. The actual clinical blood samples were tested in microfluidic SERS chip to further illustrate separation and test effectiveness of the chip. The whole blood of six cases clinical blood samples were injected into microfluidic SERS chip, the serum was separated under the optimized condition and pumped into the test chamber under the effect of negative pressure for SERS test, and the whole process could be finished within 2 min. According to the results, number 1, 2, and 3 were healthy people, and number 4, 5 and 6 were chronic renal failure, which were in conformity with the results detected by the enzyme method (Table 1). The 9
above results indicated that the separation of the whole blood and the analysis of serum creatinine by SERS detection could be completed on microfluidic SERS chip, and the test result could be used for quick auxiliary diagnosis of chronic renal failure.
4. Conclusions According to the requirement of rapid diagnosis of clinical blood, the centrifugal microfluidic technology and SERS technology were combined to design an integrated microfluidic SERS chip with array element consisted of separation-decantation cavity, mixing channels and SERS detection chamber. The SERS signal enhancement was achieved by preparing nano-Au on Ag film SERS substrate in the SERS detection chamber. Rapid separation and in situ test of serum in different blood samples could be achieved at the same time within 2 min. The high quality serum could be effectively separated when the channel vertical distance (X) between decantation and separation cavity was 2 mm, centrifugal speed 1000 rpm, and centrifugal time 100 s. The serum creatinine was selected as the test template, its characteristic peak at 678 cm
-1
could be identified effectively. The lowest identified concentration of serum
creatinine was 4.42 × 10-3 μmol mL-1 in water and 8.0 × 102 μmol mL-1 in serum. The differences are caused by the complex composition of serum. The blood samples from patients with chronic renal failure and healthy people were tested by microfluidic SERS chip method, and the results were consistent with the test data by enzymatic method. The designed microfluidic SERS chip and the established test method in this paper could be used to achieve the integration of blood separation and serum creatinine SERS rapid test. Meanwhile, multiple microfluidic SERS units were integrated on the chip, which could realize the parallel test of the same blood samples or multiple tests of different blood samples. The demand of blood samples and the cost of blood separation and detection could be effectively reduced by this method. Besides, this method was convenient to operate, could be recycled, and has potential applications in quick diagnostics.
Acknowledgements The work was financially supported by National Natural Science Foundation of China [No. 21375156], National High Technology Research and Development Program
of
China
(Ministry
of
Science
and
Technology
863
Plan)
[No.2015AA021104], Fundamental Research Funds for the Central Universities, Key 10
Project (Fund for Brain Science), [No.10611CDJXZ238826], Start-up Funding for the ‘Hundred Young-Talent Scheme’ Professorship provided by Chongqing University in China [02100011044104], Open Project form the State Key Laboratory of Transducer Technology, China [NO. SKT1705], Fundamental Research Funds for the Central Universities [NO. 106112017CDJPT120002] and Major Theme Project of Chongqing Artificial Intelligence Technology Innovation, [cstc2017rgzn-zdyfX0019].
References [1] H. Wang, Y. Liu, C. Liu, J. Huang, P. Yang, B. Liu, Microfluidic chip-based aptasensor for amplified electrochemical detection of human thrombin, Electrochemistry Communications, 12(2010) 258-61. [2] C.-C. Hong, P.-H. Chang, C.-C. Lin, C.-L. Hong, A disposable microfluidic biochip with on-chip molecularly imprinted biosensors for optical detection of anesthetic propofol, Biosensors and Bioelectronics, 25(2010) 2058-64. [3] R. Fan, O. Vermesh, A. Srivastava, B.K.H. Yen, L. Qin, H. Ahmad, et al., Integrated barcode chips for rapid, multiplexed analysis of proteins in microliter quantities of blood, Nature Biotechnology, 26(2008) 1373-8. [4] J. Steigert, M. Grumann, T. Brenner, L. Riegger, J. Harter, R. Zengerle, et al., Fully integrated whole blood testing by real-time absorption measurement on a centrifugal platform, Lab on A Chip, 6(2006) 1040. [5] I.F. Cheng, H.-C. Chang, T.-Y. Chen, C. Hu, F.-L. Yang, Rapid (<5 min) identification of pathogen in human blood by electrokinetic concentration and surface-enhanced Raman spectroscopy, Sci Rep, 3(2013) 2365. [6] A. Kamińska, E. Witkowska, K. Winkler, I. Dzięcielewski, J.L. Weyher, J. Waluk, Detection of Hepatitis B virus antigen from human blood: SERS immunoassay in a microfluidic system, Biosensors and Bioelectronics, 66(2015) 461-7. [7] X. Chen, D.F. Cui, C.C. Liu, H. Li, Microfluidic chip for blood cell separation and collection based on crossflow filtration, Sensors and Actuators B: Chemical, 130(2008) 216-21. [8] H. Jiang, X. Weng, C.H. Chon, X. Wu, D. Li, A microfluidic chip for blood plasma separation using electro-osmotic flow control, Journal of Micromechanics and Microengineering, 21(2011) 085019. [9] A. Samborski, P. Jankowski, J. grzyn, J.A. Michalski, Paw, S. owska, et al., Blood 11
diagnostics using sedimentation to extract plasma on a fully integrated point care microfluidic system, Engineering in Life Sciences, 15(2015) 333-9. [10] M. Toner, D. Irimia, Blood-on-a-chip, Annual Review of Biomedical Engineering, 7(2005) 77. [11] J.W. Martin, M.K. Nieuwoudt, M.J.T. Vargas, O.L.C. Bodley, T.S. Yohendiran, R.N. Oosterbeek, et al., Raman on a disc: high-quality Raman spectroscopy in an open channel on a centrifugal microfluidic disc, Analyst, 142(2017) 1682-8. [12] O. Durucan, T. Rindzevicius, M.S. Schmidt, M. Matteucci, A. Boisen, Nanopillar Filters for Surface-Enhanced Raman Spectroscopy, Acs Sens, 2(2017) 1400-4. [13] X. Li, W. Chen, G. Liu, W. Lu, J. Fu, Continuous-flow microfluidic blood cell sorting
for
unprocessed
whole
blood
using
surface-micromachined
microfiltration membranes, Lab on A Chip, 14(2014) 2565-75. [14] R. Gorkin, J. Park, J. Siegrist, M. Amasia, B.S. Lee, J.-M. Park, et al., Centrifugal microfluidics for biomedical applications, Lab on A Chip, 10(2010) 1758-73. [15] X.F. Chen, J.N. Kuo, Blood separation and plasma preparation on a compact disk microfluidic chip,
The 9th IEEE International Conference on Nano/Micro
Engineered and Molecular Systems (NEMS)2014, pp. 47-50. [16] M. Amasia, M. Madou, Large-volume centrifugal microfluidic device for blood plasma separation, Bioanalysis, 2(2010) 1701-10. [17] S. Pahlow, A. März, B. Seise, K. Hartmann, I. Freitag, E. Kämmer, et al., Bioanalytical application of surface- and tip-enhanced Raman spectroscopy, Engineering in Life Sciences, 12(2012) 131-43. [18] K. Hering, D. Cialla, K. Ackermann, T. Dörfer, R. Möller, H. Schneidewind, et al., SERS: a versatile tool in chemical and biochemical diagnostics, Anal Bioanal Chem, 390(2008) 113-24. [19] W. Tsuei-Lian, P. Fang-Ying, L. Hui-Hsin, H. Yung-Ta, F. Tung-Jing, C. Huihua Kenny, Surface enhanced Raman spectra in biochemical molecules and cell detection, The Second Asian and Pacific Rim Symposium on Biophotonics, 2004 APBP 20042004, pp. 57-8. [20] M.Z. Si, Y.P. Kang, R.M. Liu, Surface-enhanced Raman scattering (SERS) spectra of three kinds of azo-dye molecules on silver nanoparticles prepared by electrolysis, Applied Surface Science, 258(2012) 5533-7. [21] C. Wang, Y. Xu, C. Deng, Z. Liu, R. Wang, H. Zhao, Design and preparation of a 12
recyclable microfluidic SERS chip with integrated Au@Ag/TiO2 NTs, RSC Advances, 6(2016) 113115-22. [22] R. Wang, Y. Xu, R. Wang, C. Wang, H. Zhao, X. Zheng, et al., A microfluidic chip based on an ITO support modified with Ag-Au nanocomposites for SERS based determination of melamine, Microchimica Acta, 184(2017) 279-87. [23] L. Morelli, L. Serioli, F.A. Centorbi, C.B. Jendresen, M. Matteucci, O. Ilchenko, et al., Injection molded lab-on-a-disc platform for screening of genetically modified E. coli using liquid-liquid extraction and surface enhanced Raman scattering, Lab Chip, 18(2018) 869-77. [24] H.-Z. Zhao, Y. Xu, C.-Y. Wang, R. Wang, S.-T. Xiang, L. Chen, Design and fabrication of a microfluidic SERS chip with integrated Ag film@nanoAu, RSC Advances, 6(2016) 14105-11. [25] J.-N. Kuo, X.-F. Chen, Decanting and mixing of supernatant human blood plasma on centrifugal microfluidic platform, Microsyst Technol, 22(2016) 861-9. [26] C.-H. Shih, C.-H. Lu, W.-L. Yuan, W.-L. Chiang, C.-H. Lin, Supernatant decanting on a centrifugal platform,
Biomicrofluidics2011, p. 13414.
[27] J. Steigert, M. Grumann, T. Brenner, K. Mittenbühler, T. Nann, J. Rühe, et al., Integrated Sample Preparation, Reaction, and Detection on a High-Frequency Centrifugal Microfluidic Platform, JALA: Journal of the Association for Laboratory Automation, 10(2005) 331-41. [28] S.H. Yazdi, K.L. Giles, I.M. White, Multiplexed detection of DNA sequences using a competitive displacement assay in a microfluidic SERRS-based device, Anal Chem, 85(2013) 10605-11. [29] K. Vikram, S. Mishra, S.K. Srivastava, R.K. Singh, Low temperature Raman and DFT study of creatinine, Journal of Molecular Structure, 1012(2012) 141-50. [30] S. Feng, D. Lin, J. Lin, B. Li, Z. Huang, G. Chen, et al., Blood plasma surface-enhanced Raman spectroscopy for non-invasive optical detection of cervical cancer, Analyst, 138(2013) 3967-74.
Table 1 The Comparison of detection results by Enzyme method and microfluidic SERS chip mehod Serial
Microfluidic SERS chip mehod 13
The detection result of
Number
The peak at 678 cm-1
The detection result
enzyme method
1
None
-
-
2
None
-
-
3
None
-
-
4
Existence
+
+
5
Existence
+
+
6
Existence
+
+
(“+” represents people has the problem of chronic renal failure; “-” represents people is healthy.)
Highlights
SERS substrate was composed of nano-Au on Ag film which was prepared in-situ in the detecting chamber on the designed microchip. The special separating channels was designed for blood separation on the chip. The separated serum was directly imported into the detecting chamber for SERS detection. An integrated microfluidic SERS chip with multifunction of separation and SERS detection was applied to detect rapidly the clinical blood within 2 min.
14
15
16
17
18
PII: DOI: Reference:
S0039-9140(18)31164-0 https://doi.org/10.1016/j.talanta.2018.11.014 TAL19247
To appear in: Talanta Received date: 28 July 2018 Revised date: 26 October 2018 Accepted date: 5 November 2018 Cite this article as: Xi Su, Yi Xu, Huazhou Zhao, Shunbo Li and Li Chen, Design and preparation of centrifugal microfluidic chip integrated with SERS detection for rapid diagnostics, Talanta, https://doi.org/10.1016/j.talanta.2018.11.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Design and preparation of centrifugal microfluidic chip integrated with SERS detection for rapid diagnostics Xi Sub,c, Yi Xua,b,c,*, Huazhou Zhaob,c, Shunbo Lia,c, Li Chena,c,* a
Key Disciplines Laboratory of Novel Micro-Nano Devices and System
Technology, Key Laboratory of Optoelectronic Technology and Systems, Ministry of Education, School of Optoelectronics Engineering, Chongqing University, Chongqing 400044, China b
School of Chemistry and Chemical Engineering, Chongqing University,
Chongqing 400044, China c
International R & D center of Micro-nano Systems and New Materials
Technology, Chongqing 400044, China [email protected] [email protected]
*Corresponding author: Abstract A microfluidic SERS chip integrated with blood separation and in-situ detection was designed and fabricated for the rapid detection of clinical blood samples. Each functional unit in the microfluidic SERS chip consist of separation-decantation cavity based on centrifugal separation principle, mixing channels and SERS detection chamber built with integrated nano-Au on Ag film microstructure. The serum creatinine was selected as a typical sample to demonstrate the capability of microfluidic SERS chip. It was found that the creatinine SERS characteristic peaks at 678 cm-1 can be effectively identified and the detection limit could be as low as 4.42 × 10-3 μmol mL-1 in water. The blood samples were also tested in microfluidic SERS chip. The whole separation and test process could be completed within 2 min, which is a significant improvement in the field of creatinine detection. The whole blood of six cases clinical blood samples were also tested, and the results were consistent with the enzymatic results. The developed microfluidic SERS chip has advantages including reduction of the required quantity of blood sample, reusable and easy to operate. It is expected to provide a new method for rapid diagnostics.
1
Graphical Abstract
Key words: Surface enhanced Raman spectroscopy (SERS), Ag film@nano-Au substrate, centrifugal microfluidic chip, blood separation, rapid diagnostics
1. Introduction The micro-Total analysis system (µTAS) developed very rapidly in the field of point of care in last decades due to its unique advantages such as less sample volume, fast detection, high throughput, low contamination and easy integration ect. Lots of analytical methods including electrochemistry[1], UV-vis[2], fluorescence[3] and Raman/SERS[4-6] have been applied in the whole blood test, after the pretreament of serum extraction. Compared to conventional techniques, microfluidic analytical method is more popular in clinical blood test due to the high efficiency and elimination of large scale apartus, such as LC、HPLC、LC-MS、GC-MS and so on. The serum extraction is a crucial step in whole blood test since the blood cells could interfere with the detection of the target molecules. The common methods for the separation of blood in microfluidic chip include physical method based on particle size[7], electrophoresis[8] and centrifugation separation[9-12]. The separation based on particle size has the problem of channel blockage by blood cells, as well as the low separation efficiency [7, 13]. Electrophoretic separation technology has good analytical efficiency, however, it has inevitable cell damage and hemolysis phenomenon caused by the high driving voltage, and it is difficult to obtain the 2
original and stable serum samples[8]. Existing studies have shown that the centrifugal microfluidic technology can obtain high quality separation serum, based on the separation principle that the mass density of each blood component is different[14]. Chen et al[15] designed an integrated chip system with separation-decantation microchannel network to separate serum from the whole blood. However, only the diluted blood sample could be separated to obtain high quality serum in this chip, and the obtained serum used for detection was less than 1 μL, which was below the required sample amount. Amasia et al[16] presented a new device with larger centrifugal disc for serum separation within about 5 min, which could deal with 2 mL of undiluted blood samples at a time and obtain high quality serum. The potential and advantages of centrifugal microfluidic technology in blood separation were showed by above researches. However, a mature microfluidic chip integrated with blood separation and detection units needs to be studied and expanded. Surface Enhanced Raman spectrum (SERS) can greatly enhance Raman signal due to the strong interactions between moleculars and the surface of rough metal nanostructures[17]. SERS, with features of high sensitivity, non-interference by water and transparent packaging material, has a distinct advantage on fast identification and analysis of complex blood samples [17-19]. Gold and silver nanoparticles were the most widely used metal among SERS materials. The Ag nanostructure has a strong SERS effect[20]. However, the Ag NPs were very active, and the surface could be easily oxidized, while the Au NPs have good stability and biocompatibility. The oxidation of the Ag NPs film can be prevented, if a layer of Au NPs were assembled on the surface of the silver NPs film, and the interaction between the substrate and biological samples can be enhanced[21, 22]. Therefore, microfluidic chip technology and SERS technology were combined to form integrated microfluidic SERS chip to realize high efficient separation and analysis of blood, which is the new trend and research hotspot of developing the clinical rapid detection field[23]. According to the demand of rapid diagnostics, an integrated microfluidic SERS chip including blood separation and SERS in situ detection was designed and prepared. The centrifugal microfluidic technology and SERS technology were well organized
in
the
developed
microfluidic
SERS
chip,
which
consist
of
separating-decantation cavity, mixing channel and detection cavity. Nano-Au on Ag film SERS substrate was designed and prepared in the detection cavity to improve the sensitivity. The serum creatinine was selected as the test object. The functions of 3
separation and detection in microfluidic SERS chip were selected and optimized, and a new method for serum test was established. The reliability of the developed device was proved using blood samples from healthy person and patients with chronic renal failure. A new method for the diagnosis of clinical nephropathy was provided by the established testing method used microfluidic SERS chip.
2. Materials and methods 2.1 Materials Hydrogen tetrachloroauric acid (HAuCl4·4H2O, AR) was purchased from Sinopharm chemical reagent Co. Ltd (Shang Hai, China). Silver nitrate (AgNO3, AR) was purchased from Aladdin (Shang Hai, China). Stannous chloride (SnCl2, AR), Trisodium citrate (AR), glucose (AR), Ammonia water (NH3·H2O, AR), Hydrogen peroxide (H2O2, 30%), Concentrated sulfuric acid(H2SO4, 98%),
Concentrated
hydrochloric acid (HCl, AR), Concentrated nitric acid(HNO3, AR) were purchased from Chong Qing Chuandong Chemical (Group) CO., LTD. The SU-8 photoresist and developer was purchased from Microchem, and poly(dimethylsiloxane) (PDMS) from Dow Corning (Sylgard 184). Blood from healthy people was obtained from Hospital of Chongqing University, blood from patients with chronic renal failure was provided by Southwestern Hospital. 2.2 Sample preparation Creatinine samples with concentrations of 8.84 × 103 μmol mL-1, 1.18 × 104 μmol mL-1 and 1.33 × 104 μmol mL-1 were prepared by dissolving 0.1000g、0.1332g、 0.1500g standard creatinine powder in deionized water respectively. Then, the solution with 8.84 × 103 μmol mL-1 creatinine was diluted into a series concentration of creatinine solution from 4.42 × 10-3 to 44.2 μmol mL-1. The blood from healthy people with creatinine were prepared by mixing the blood with the as-prepared creatinine solutions (1.6 × 103, 3.2 × 103, 4.8 × 103, 8.0 × 103, 1.12 × 104, 1.28 × 104 μmol mL-1) with 1:1 ratio to obtain the desired concentrations. 2.3 Instrumentations Confocal Raman (HORIBA LABRAM, France) equipped with a 633 nm He–Ne laser as excitation source was used for detection. The power of the excitation source was about 17 mW, and the spectral resolution of this instrument was 2 cm-1. The 600 gr•mm-1 gratings and a neutral density filter of 10% were used during the detection. The Raman spectra were collected through a 50× microscope objective lens (NA 0.5) 4
and acquisition time of 4 s was used. The blood separation was realized by SJT-1 Spinner purchased from Shanghai Xueze Optical Machinery Co., Ltd. The surface morphology of Nano-Au on Ag film substrate were characterized using scanning electron microscopy (SEM, Hitachi 7800F system, Japan). The samples were pumped into the chip by Flow injection pump (HARWARD，USA). 2.4 Preparation of Microfluidic SERS chip The Microfluidic SERS chip consists of two parts: glass substrate and PDMS cover.
Glass
substrate
with
microchannels
was
prepared
using
standard
photolithography and wet etching technology. The cover was prepared by mixing PDMS prepolymer and curing agent, pouring onto a cleaned and smooth glass plate, and cured for 2 h at 90 ℃. Then, the inlets and outlets of chip were punched for tubing connection. The prepared glass substrate was merged in hot piranha solution for 30 min to make the surface hydroxylation, then rinsed by deionized water and ethanol. The PDMS cover was cleaned by means of ultrasonic cleaning for 5 min in the deionized water and ethanol, and dried by nitrogen. Glass substrate and PDMS cover were assembled, and placed under 75 ℃ for 10 min to strengthen the bonding force between them. Finally, bonding based on physical function between the substrate and cover plate was realized. The nano-Au on Ag film SERS substrate was prepared by self-assembly method.[24] Firstly, SnCl2 solution (0.02 wt%) was introduced in the SERS detection chamber to activate the glass substrate. The newly prepared silver-ammonia solution and 5% glucose solution were mixed by the volume ratio of 1:5, then the mixture was pumped to the SERS detection chamber in a 60 ℃ water bath for 10 min to form shining silver mirror. In order to obtain self-assembled AuNPs on the Ag film, the SERS detection chamber was immersed in 1% cationic polymer PDDA for 30 min, rinsed by DI water, and the gold sol (Hydrogen tetrachloroauric acid) was injected to react for 6 h. It was noted that gold sol was renewed every 30 min. The microfluidic SERS chip integrated with nano-Au on Ag film SERS was prepared. 2.5 Microfluidic SERS chip test 3 μL blood was pumped into each separation chamber of microfluidic SERS chip for blood separation test. The chip size parameter, rotation speed and time were optimized after observing clear serum separation in the separation-decantation cavity 5
in the device. The standard creatinine powder and 8.84 × 103 μmol mL-1 creatinine solution were tested by the microfluidic SERS chip to determine characteristic peak of creatinine molecular, deionized water and serum from healthy people were used as background signal. Different concentrations of creatinine solution or healthy blood contained different concentrations of creatinine were tested in microfluidic SERS chip. The detection effect was evaluated by averaging spectra from three times test of each sample. The serum SERS spectra were used to establish the relationship between the creatinine concentration and characteristic peak intensity. In addition, six clinical blood samples were tested by the microfluidic SERS chip method respectively. The conventional enzyme method was also performed for comparison between two methods.
3. Results and discussion 3.1 The microfluidic SERS chip The designed and fabricated microfluidic SERS chip with double function of separation and SERS in situ detection consists of three parts: separation-decantation cavity, mixing channel and SERS detection chamber (Fig. 1). There were four pairs (8 units) of channels symmetrically distributed on the device. The size of the structure was shown in Fig. 1, and the depths were 80 μm for all the channels. When microfluidic SERS chip was rotating, blood sample in the separation cavity was affected by three kinds of force - centrifugal force (Fw) produced by the system rotation, centripetal force (Fc) by the systems and fluid velocity and centrifugal force (Fr) by the curvature of the micro-channels[25]. The different densities between the blood cells and serum brought out the different net forces. Cells with larger density experienced larger net force and deposited on the bottom of the separation chamber. On the contrary, serum with smaller density was located in the upper part. By controlling the centrifugal rate, the separated serum was easily imported into the decantation cavity through the micro-channel between the separation cavity and decantation cavity, so that the blood cells and serum were separated [26, 27]. The hole on the decantation cavity could balance the pressure of atmosphere. The serpentine mixing micro-channels extended to the direction of the center, which connected the decantation cavity and SERS detection chamber. The separated 6
serum could be imported into the SERS detection chamber by negative pressure through the external pipe of microfluidic SERS chip’s center. The mixing channels were beneficial to realize a variety of samples mixing quickly and sufficiently under the condition of micro-scale. In particular, the mixing efficiency could be optimized by adjusting the length of the channel[28]. In order to achieve fully mix, the length of the serpentine channel was set to about 36,840 μm and the channel width was 200 μm. Negative pressure was applied at the end of the detection chamber to direct the serum to the decantation cavity, and then flow into the detection chamber integrated with nano-Au on Ag film substrate for SERS detection. Owing to the design of separation chamber, there is no interference of Raman signal caused by the blood cells. The sensitivity of SERS detection signal could be improved effectively through the synergistic effect among Au nanoparticles and between Au nanoparticles and Ag film.[21] The key and difficulty in microfluidic SERS chip was the integration of nano-Au on Ag film on the glass, so self-assembly method, which was easy to carry out and realize integration in microchannels, was introduced. A layer of Ag nano film was deposited on the surface of the detection chamber by self-assembly method described in part 2.3, the SEM image of Ag film showed that islands of silver particles were formed after 10 min of the silver mirror reaction (Fig. 2a). Then Au nanoparticles were assembled on the Ag film, and the Au nanoparticles（~100 nm） was uniformly distributed on the surface of Ag islands (Fig. 2b), and finally the nano-Au on Ag film SERS substrate was integrated in the SERS detection chamber successfully. 3.2 Optimization of the separation process Due to the total volume of the separation cavity was 2.24 μL by calculating. 3 μL of blood was selected as the optimized amount for processing. If the amount of blood sample was excessive and beyond the capacity below the liquid outlet, blood cells might flow into the mixing channels, serum and blood cells could not be separated well. On the contrary, the serum and blood cells would be remained in the separation cavity and the serum could not be separated into the decantation cavity. The initial width (X in Fig. 1) of the micro-channel connecting the separation and decantation cavity is an important parameter which determines whether the serum above the separation chamber could flow smoothly into the decantation cavity. In order to optimize the separation of microfluidic SERS chip, the vertical distance X was optimized. So as to select the most appropriate width, three kinds of width were 7
designed. X was 1 mm in 1 and 5 unit, 2 mm in 2, 4, 6 and 8 unit, and 3 mm in 3 and 7 structure unit. The centrifugal speed was set to 1000 rpm for 100 s to ensure that the blood cells were not ruptured after loading blood samples. The separation results were showed in Fig. 3. The blood in unit 1 and 5 were stayed in the separation cavity (Fig. 3a), and with the increase of the spin time, serum - blood cells were distributed in two layers in the separation cavity, but the serum could not enter the decantation cavity. The serum - blood cells in unit 2 and 6 were separated well (Fig. 3b), and serum with a spot of blood cells flowed into the decantation cavity. The small amount of blood cells will stay on the bottom of the decantation cavity, so there was no interference to the test. In unit 3 and 7, a large number of blood cells entered into the decantation cavity (Fig. 3 c), and the separation effect was poor. It was not possible for the subsequent serum test. Therefore, 2 mm was optimum for the initial vertical distance (X) of the channel between the separation cavity and the decantation cavity. 3 μL blood was tested in microfluidic SERS chip for spin time of 20, 40, 60, 80, 100 s to optimize the separation time (Fig. 4 a-e). As the separation time was increased, there were more and more blood flowed into the separation cavity from the input port. When the separation time was 80 s, the blood was nearly full in the separation cavity. And when 100 s was applied, all of the 3 μL blood filled into the separation cavity, the blood serum and blood cells were separated perfectly, and the vast majority of serum and a small number of blood cells flowed into the decantation cavity for the subsequent SERS test of serum. Therefore, the optimized centrifugal separation time was identified to be 100 s. 3.3 Performance of the SERS detection on the microfluidic chip The creatinine standard powder was tested with Raman to confirm the characteristic peak of creatinine detected by microfluidic SERS chip, and the results were shown in Fig. 5. The characteristic peak at 678 cm -1 comes from shear vibration of the ring plane, stretching vibration of C - NH2, the ring and C - O. It was used for the trademark peak of creatinine [29]. Deionized water and creatinine aqueous solution with concentration of 8.84 x 103 μmol mL-1 were tested in microfluidic SERS chip integrated with nano-Au on Ag film substrate, and the SERS signals were collected (Fig. 6). The background peaks of the substrate were mainly concentrated at 784 cm -1, 715 cm -1, 1001 cm -1, 1395 cm -1, 1240 cm -1, 1544 cm -1, and 1456 cm -1, and most of the peaks were weak, so the characteristic peak of creatinine would not be interfered by the background peaks. The above results illustrated that the microfluidic 8
SERS chip could be used for creatinine SERS test. The detection sensitivity in microfluidic SERS chip for creatinine molecule was also evaluated. creatinine solution with concentration of 4.42 × 10-3, 4.42 × 10-2, 0.442, 4.42 and 44.2 μmol mL-1 were tested in microfluidic SERS chip respectively, the results were shown in Fig. 7. With the reduction of the creatinine concentration, the SERS intensity at the characteristic peak of creatinine (678 cm -1) was gradually reducing. The characteristic peak could still be identified when the concentration of creatinine was as low as 4.42 × 10-3 μmol mL-1, suggesting that the detection limit of creatinine using the as-prepared nano-Au on Ag film SERS substrate was 4.42 × 10-3 μmol mL-1. 3.4 Serum creatinine detection by microfluidic SERS chip In order to verify that microfluidic SERS chip could be used for serum creatinine detection, the healthy human blood and the healthy human blood with different concentrations of creatinine were tested. The separated serums were pumped into the detection chamber for SERS test. The SERS sepctra of the separated creatinine were collected as shown in Fig. 8. It was illustrated that the characteristic peak of creatinine at 678 cm-1 was observed, and the peak intensity was increased with the increase of creatinine concentration, but there was no obvious linear relationship between peak intesity and creatinine concentration. The detection limit of creatinine using the as-prepared nano-Au on Ag film SERS substrate was 8.0 × 102 μmol mL-1. It was noted that the peak strength of the characteristic peak of creatinine was affected by the background peak of nano-Au on Ag film substrate and the complex serum composition, resulting in a higher detection limit for serum creatinine detected by this method[30]. The precise quantitative analysis of serum creatinine by this microfluidic SERS chip was difficult in the certain degree, but the chip could be used for auxiliary diagnosis of chronic renal failure. The actual clinical blood samples were tested in microfluidic SERS chip to further illustrate separation and test effectiveness of the chip. The whole blood of six cases clinical blood samples were injected into microfluidic SERS chip, the serum was separated under the optimized condition and pumped into the test chamber under the effect of negative pressure for SERS test, and the whole process could be finished within 2 min. According to the results, number 1, 2, and 3 were healthy people, and number 4, 5 and 6 were chronic renal failure, which were in conformity with the results detected by the enzyme method (Table 1). The 9
above results indicated that the separation of the whole blood and the analysis of serum creatinine by SERS detection could be completed on microfluidic SERS chip, and the test result could be used for quick auxiliary diagnosis of chronic renal failure.
4. Conclusions According to the requirement of rapid diagnosis of clinical blood, the centrifugal microfluidic technology and SERS technology were combined to design an integrated microfluidic SERS chip with array element consisted of separation-decantation cavity, mixing channels and SERS detection chamber. The SERS signal enhancement was achieved by preparing nano-Au on Ag film SERS substrate in the SERS detection chamber. Rapid separation and in situ test of serum in different blood samples could be achieved at the same time within 2 min. The high quality serum could be effectively separated when the channel vertical distance (X) between decantation and separation cavity was 2 mm, centrifugal speed 1000 rpm, and centrifugal time 100 s. The serum creatinine was selected as the test template, its characteristic peak at 678 cm
-1
could be identified effectively. The lowest identified concentration of serum
creatinine was 4.42 × 10-3 μmol mL-1 in water and 8.0 × 102 μmol mL-1 in serum. The differences are caused by the complex composition of serum. The blood samples from patients with chronic renal failure and healthy people were tested by microfluidic SERS chip method, and the results were consistent with the test data by enzymatic method. The designed microfluidic SERS chip and the established test method in this paper could be used to achieve the integration of blood separation and serum creatinine SERS rapid test. Meanwhile, multiple microfluidic SERS units were integrated on the chip, which could realize the parallel test of the same blood samples or multiple tests of different blood samples. The demand of blood samples and the cost of blood separation and detection could be effectively reduced by this method. Besides, this method was convenient to operate, could be recycled, and has potential applications in quick diagnostics.
Acknowledgements The work was financially supported by National Natural Science Foundation of China [No. 21375156], National High Technology Research and Development Program
of
China
(Ministry
of
Science
and
Technology
863
Plan)
[No.2015AA021104], Fundamental Research Funds for the Central Universities, Key 10
Project (Fund for Brain Science), [No.10611CDJXZ238826], Start-up Funding for the ‘Hundred Young-Talent Scheme’ Professorship provided by Chongqing University in China [02100011044104], Open Project form the State Key Laboratory of Transducer Technology, China [NO. SKT1705], Fundamental Research Funds for the Central Universities [NO. 106112017CDJPT120002] and Major Theme Project of Chongqing Artificial Intelligence Technology Innovation, [cstc2017rgzn-zdyfX0019].
References [1] H. Wang, Y. Liu, C. Liu, J. Huang, P. Yang, B. Liu, Microfluidic chip-based aptasensor for amplified electrochemical detection of human thrombin, Electrochemistry Communications, 12(2010) 258-61. [2] C.-C. Hong, P.-H. Chang, C.-C. Lin, C.-L. Hong, A disposable microfluidic biochip with on-chip molecularly imprinted biosensors for optical detection of anesthetic propofol, Biosensors and Bioelectronics, 25(2010) 2058-64. [3] R. Fan, O. Vermesh, A. Srivastava, B.K.H. Yen, L. Qin, H. Ahmad, et al., Integrated barcode chips for rapid, multiplexed analysis of proteins in microliter quantities of blood, Nature Biotechnology, 26(2008) 1373-8. [4] J. Steigert, M. Grumann, T. Brenner, L. Riegger, J. Harter, R. Zengerle, et al., Fully integrated whole blood testing by real-time absorption measurement on a centrifugal platform, Lab on A Chip, 6(2006) 1040. [5] I.F. Cheng, H.-C. Chang, T.-Y. Chen, C. Hu, F.-L. Yang, Rapid (<5 min) identification of pathogen in human blood by electrokinetic concentration and surface-enhanced Raman spectroscopy, Sci Rep, 3(2013) 2365. [6] A. Kamińska, E. Witkowska, K. Winkler, I. Dzięcielewski, J.L. Weyher, J. Waluk, Detection of Hepatitis B virus antigen from human blood: SERS immunoassay in a microfluidic system, Biosensors and Bioelectronics, 66(2015) 461-7. [7] X. Chen, D.F. Cui, C.C. Liu, H. Li, Microfluidic chip for blood cell separation and collection based on crossflow filtration, Sensors and Actuators B: Chemical, 130(2008) 216-21. [8] H. Jiang, X. Weng, C.H. Chon, X. Wu, D. Li, A microfluidic chip for blood plasma separation using electro-osmotic flow control, Journal of Micromechanics and Microengineering, 21(2011) 085019. [9] A. Samborski, P. Jankowski, J. grzyn, J.A. Michalski, Paw, S. owska, et al., Blood 11
diagnostics using sedimentation to extract plasma on a fully integrated point care microfluidic system, Engineering in Life Sciences, 15(2015) 333-9. [10] M. Toner, D. Irimia, Blood-on-a-chip, Annual Review of Biomedical Engineering, 7(2005) 77. [11] J.W. Martin, M.K. Nieuwoudt, M.J.T. Vargas, O.L.C. Bodley, T.S. Yohendiran, R.N. Oosterbeek, et al., Raman on a disc: high-quality Raman spectroscopy in an open channel on a centrifugal microfluidic disc, Analyst, 142(2017) 1682-8. [12] O. Durucan, T. Rindzevicius, M.S. Schmidt, M. Matteucci, A. Boisen, Nanopillar Filters for Surface-Enhanced Raman Spectroscopy, Acs Sens, 2(2017) 1400-4. [13] X. Li, W. Chen, G. Liu, W. Lu, J. Fu, Continuous-flow microfluidic blood cell sorting
for
unprocessed
whole
blood
using
surface-micromachined
microfiltration membranes, Lab on A Chip, 14(2014) 2565-75. [14] R. Gorkin, J. Park, J. Siegrist, M. Amasia, B.S. Lee, J.-M. Park, et al., Centrifugal microfluidics for biomedical applications, Lab on A Chip, 10(2010) 1758-73. [15] X.F. Chen, J.N. Kuo, Blood separation and plasma preparation on a compact disk microfluidic chip,
The 9th IEEE International Conference on Nano/Micro
Engineered and Molecular Systems (NEMS)2014, pp. 47-50. [16] M. Amasia, M. Madou, Large-volume centrifugal microfluidic device for blood plasma separation, Bioanalysis, 2(2010) 1701-10. [17] S. Pahlow, A. März, B. Seise, K. Hartmann, I. Freitag, E. Kämmer, et al., Bioanalytical application of surface- and tip-enhanced Raman spectroscopy, Engineering in Life Sciences, 12(2012) 131-43. [18] K. Hering, D. Cialla, K. Ackermann, T. Dörfer, R. Möller, H. Schneidewind, et al., SERS: a versatile tool in chemical and biochemical diagnostics, Anal Bioanal Chem, 390(2008) 113-24. [19] W. Tsuei-Lian, P. Fang-Ying, L. Hui-Hsin, H. Yung-Ta, F. Tung-Jing, C. Huihua Kenny, Surface enhanced Raman spectra in biochemical molecules and cell detection, The Second Asian and Pacific Rim Symposium on Biophotonics, 2004 APBP 20042004, pp. 57-8. [20] M.Z. Si, Y.P. Kang, R.M. Liu, Surface-enhanced Raman scattering (SERS) spectra of three kinds of azo-dye molecules on silver nanoparticles prepared by electrolysis, Applied Surface Science, 258(2012) 5533-7. [21] C. Wang, Y. Xu, C. Deng, Z. Liu, R. Wang, H. Zhao, Design and preparation of a 12
recyclable microfluidic SERS chip with integrated Au@Ag/TiO2 NTs, RSC Advances, 6(2016) 113115-22. [22] R. Wang, Y. Xu, R. Wang, C. Wang, H. Zhao, X. Zheng, et al., A microfluidic chip based on an ITO support modified with Ag-Au nanocomposites for SERS based determination of melamine, Microchimica Acta, 184(2017) 279-87. [23] L. Morelli, L. Serioli, F.A. Centorbi, C.B. Jendresen, M. Matteucci, O. Ilchenko, et al., Injection molded lab-on-a-disc platform for screening of genetically modified E. coli using liquid-liquid extraction and surface enhanced Raman scattering, Lab Chip, 18(2018) 869-77. [24] H.-Z. Zhao, Y. Xu, C.-Y. Wang, R. Wang, S.-T. Xiang, L. Chen, Design and fabrication of a microfluidic SERS chip with integrated Ag film@nanoAu, RSC Advances, 6(2016) 14105-11. [25] J.-N. Kuo, X.-F. Chen, Decanting and mixing of supernatant human blood plasma on centrifugal microfluidic platform, Microsyst Technol, 22(2016) 861-9. [26] C.-H. Shih, C.-H. Lu, W.-L. Yuan, W.-L. Chiang, C.-H. Lin, Supernatant decanting on a centrifugal platform,
Biomicrofluidics2011, p. 13414.
[27] J. Steigert, M. Grumann, T. Brenner, K. Mittenbühler, T. Nann, J. Rühe, et al., Integrated Sample Preparation, Reaction, and Detection on a High-Frequency Centrifugal Microfluidic Platform, JALA: Journal of the Association for Laboratory Automation, 10(2005) 331-41. [28] S.H. Yazdi, K.L. Giles, I.M. White, Multiplexed detection of DNA sequences using a competitive displacement assay in a microfluidic SERRS-based device, Anal Chem, 85(2013) 10605-11. [29] K. Vikram, S. Mishra, S.K. Srivastava, R.K. Singh, Low temperature Raman and DFT study of creatinine, Journal of Molecular Structure, 1012(2012) 141-50. [30] S. Feng, D. Lin, J. Lin, B. Li, Z. Huang, G. Chen, et al., Blood plasma surface-enhanced Raman spectroscopy for non-invasive optical detection of cervical cancer, Analyst, 138(2013) 3967-74.
Table 1 The Comparison of detection results by Enzyme method and microfluidic SERS chip mehod Serial
Microfluidic SERS chip mehod 13
The detection result of
Number
The peak at 678 cm-1
The detection result
enzyme method
1
None
-
-
2
None
-
-
3
None
-
-
4
Existence
+
+
5
Existence
+
+
6
Existence
+
+
(“+” represents people has the problem of chronic renal failure; “-” represents people is healthy.)
Highlights
SERS substrate was composed of nano-Au on Ag film which was prepared in-situ in the detecting chamber on the designed microchip. The special separating channels was designed for blood separation on the chip. The separated serum was directly imported into the detecting chamber for SERS detection. An integrated microfluidic SERS chip with multifunction of separation and SERS detection was applied to detect rapidly the clinical blood within 2 min.
14
15
16
17
18