| Microfluidic technology has been developed rapidly because the devices are largely biocompatible. It requires small sample volumes,creates small volumes of biohazard waste and enables faster analyses.Surface enhanced Raman spectrum(SERS) has the significant advantages in minimal sample preparation. It has extremely high sensitivity and ease of operation.It has been proven to be highly compatible with microfluidics.So it has raisedcomprehensive concern in the field of blood analysis. Unfortunately, little research about blood detection in microfluidic SERS chip has been studied. As we know, the constituent of blood is complex, which may result in the overlapping of SERS spectrum. Therefore, a SERS substrate with good reproducibility and high sensitivity is definitely required. If it is obtained and can be integrated in microfluidic chip fittedly, the amount of blood sample will be greatly reduced, the integration of separation and detection of blood constituent will come true, and the blood related diseases will diagnosed rapidly.The main research and the results of the paper are as follows:(1)The microfluidic chip for blood separation was designed.Based on it, blood cells and serum was effectively separated and their structures were detected and analyzed successfully by Raman spectroscopy. The differences of densitybetween blood cells and serum caused different centrifugal force under the same condition. According to it, a round PDMS-glass microfluidic SERS chip which consisted of separate-decanting structure, mixing pipeline and SERS detection area was designed. Eight similar units were distributed symmetrically by two.Separation-decanting structure located far away from the the center of the circle, which was helpful to strengthen the centrifugal force. Different vertical distances(X) between separation cavity and decanting cavity were settled as 1mm, 2mm, 3mm separately.The best separation and decanting effect was obtained when X was 2mm at 1000 rpm of centrifugal speed for 90 s. Compared to the in-situ Raman signals of blood cell in open system, the baseline drift caused by fluorescence background of blood cell had been significantly decreased in microfluidic chip. Furthermore, the Raman characteristic peaks of blood cell were more enrich and the peakwas stronger. It was good for assign the Raman characteristic peaks of blood cell. Blood cells of 15 healthy persons and 20 chronic renal failure(CRF) patients have been separated and detected by regular Raman spectroscopy. The results demonstrated that all of the Raman intensity ratios of healthy people to peak belong to tyrosine at 1618 cm-1 were stronger than that of CRF patients at756 cm-1,1004 cm-1,1122 cm-1,1226 cm-1,1550 cm-1,1640 cm-1.This was coincident with the phenonmenon ofdifferent degree ofanemia among CRF patients. The Raman intensity ratio of CRF patients totyrosine at 1618 cm-1 were lower than that of healthy people at 1004cm-1 indicated that the concentration of phenylalanine of CRF patients was lower than that of healthy people. While the finger print of human serum could not be obtained by regular Raman spectroscopy.(2)On the basis of the outstanding plasma characteristic of gold and silver, a new type of SERS substrate of Ag film@nano Au was designed and fabricated successfullyin a “sandwhich” microfluidic chip. Based on it, the serum was analyzed by SERS method.Ag film@nano Au was integrated in “sandwich” microfluidic chip through self-assemble and chemical plating method to obtainthe microfluidic SERS chip. Firstly, the prepared condition of Ag film@nano Au was optimizedwith Rodanmin 6G as the probe molecule.On the basis of that, the efficiency of the as-prepared microfluidic SERS chip and theSERS detection of blood serum were analysized.The optimized SERS effect was observed when the PDDA concentration was 0.01% and the chemical plating time was 4min. Compared to single gold and silver SERS substrate, the composite of Ag film@nano Au integrated in the microfluidic channel resulted in the peak intensityof 1507 cm-1 increasing 3-5 times during the SERSdetection of Rhodamine 6G(R6G).The strongest SERS effect was observed when the laser was focused on the interface of sample and SERS substrate.The SERS intensity of R6 G decreased 2.5 times and 2.1 times when PDMS or glass was used as cover plate separately.With 1mm of PDMS in thickness as cover plate, 10 nM of R6 G could still be detected in the as-prepared microfluidic chip and the enhancement factor(EF)was 3.8×105. A relative standard deviation(RSD) of approximately 10% was derived in the microfluidic SERS chip prepared indifferent batch.The as-prepared microfluidic SERS chip was applied to the detection of human serum. The characteristic peak of human serum has been enhanced obviously, which illustrated that the microfluidic SERS chip can be used in the research of serum as well as its components probably.(3)Base on the microfluidic SERS chip integrated with Ag film@nano Au, the analysis of creatinine in human serum was conducted and the preliminary determination about the concentration of creatinine in the serum of chronic renal failure(CRF) patients was realized.The detection limit of creatinine solution was 5×10-3mg/dlin themicrofluidic SERS chip fabricated by self-assembly and chemical plating method.Unfortunately, the effective SERS signal of 20.0-20.5 mg/dl of creatinine in serum could not be obtained.The preparation conditions of Ag film@nano Au SERS substrate were improved through self-assemble method. The best SERS effect was derived from 1% concentration of PDDA and 10 nm gold nanoparticle. The detection limit of creatinine solution in the improved microfluidic SERS chip was 5×10-5mg/dl, which decreased two orders than the pre-microfluidic SERS chip. Spiked with different concentrations of creatinine, human serum was detected in the improved microfluidic chip, and the result demonstrated that the 678cm-1 of creatinine can be recognized easily as its concentration above 10.0 mg/dl. If the concentration of creatinine in serum was lower than 10.0 mg/dl, it could not be recognized.The improved Ag film@nano Au SERS substrate was integrated in the detection area of blood separation chip and blood of typical CRF patients were separated. Then the derived serums were detected by SERS. The whole process needs less than 2 min.The results indicated that the creatinine levels detected by SERS were consistent with the results of enzymatic test.The microfluidic SERS chip for blood separation can quickly realized the blood separation and the SERS detection of creatinine in serum. |
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