Integrated Microfluidic Biochip with Nanocoating Self-assembled Fiber-optic Sensor Ming-jie Yin, Bo-bo Huang, A. Ping Zhang, Hwa-Yaw Tam, and Xue-song Ye Abstract— A microfluidic biochip integrated with highly sensitive fiber-optic glucose biosensor is presented. With the layer-bylayer self-assembly technology, poly (ethylenimine) (PEI), poly (acrylic acid) (PAA) and glucose oxidase (GOx) multilayers were deposited on the surface of a long-period fiber grating for sensing of glucose. Experimental results reveal that the biochip can achieve ultra-low detection limit (1 nM) and is very promising for diabetes mellitus detection. Keywords—microfluidic biochip, self-assembled glucose biosensor, long period grating
nanocoating,
nanometer scale [5, 6]. In addition, it was reported that LbL multilayer sensing film could overcome the loss of biomolecular activity [7]. Thus, in this study, we will use the LbL self-assembly technique to prepare (PEI/PAA)9(PEI/GOx)1 multilayer sensing films, and a highly sensitive long period fiber grating (LPG) sensor will be integrated to measure the induced refractive-index change of the sensing film. The experimental results will show that the microfluidic biochip can detect the concentration of GO as low as 1 nM. II. EXPERIMENTAL
I. INTRODUCTION Diabetes mellitus is a kind of worldwide health problem, and will induce many different kinds of diseases, e.g. heart disease, kidney failure, and blindness [1]. The main causes of the diabetes mellitus are insulin deficiency and hyperglycemia in human body. These two parameters can be reflected by blood glucose (GO) concentration higher or lower than the normal range. Therefore, it has become extremely important to detect the concentration of GO in human body for diagnostic of diabetes mellitus. Electrochemical glucose enzyme electrodes are currently the most popular GO biosensors since the first demonstration by Clark and Lyons in 1962 [2]. However, some endogenous reducing species existing in the blood, such as ascorbic and uric acids and some drugs (e.g., acetaminophen), are electroactive [3] and will lead to electroactive interferences. Moreover, a relatively large volume of bloods are usually required for electrochemical test. To solve the above problems, we report an optical fiber integrated GO biochip. As the thickness of sensing film plays a key role in fiber-optic sensors fabrication [4], layer-by-layer (LbL) self-assembly technique is chosen for preparation of the sensing film because of its flexible controllability and tunability of material properties and architecture at the
*This work was partially supported by Hong Kong RGC GRF (Grant No.: PolyU 152211/14E) and the Joint Supervision Scheme (Project Code: GSB05). M.J. Yin, B.B. Huang, A. P. Zhang, and H.-Y. Tam are with Photonics Research Center, Department of Electrical Engineering, The Hong Kong Polytechnic University, Hong Kong SAR, China (corresponding author: +852-34003336; Fax: +852-23301544; e-mail:
[email protected],
[email protected]). B. B. Huang and X. S. Ye are with Biosensor National Special Laboratory, College of Biomedical Engineering and Instrument Science, Zhejiang University, Hangzhou, 310027, China
Poly (ethylenimine) (PEI, Mw =750 000 g mol-1, 50wt% aqueous solution), poly (acrylic acid) (PAA, Mw= 100 000 g mol-1, 35 wt% aqueous solution) and glucose oxidase (GOx) (149800 U/g) were purchased from Sigma-Aldrich. Glucose anhydrous was purchased from Shenzhen Chemical Reagent Company. All inorganic materials (NaCl, H2SO4, H2O2, HCl and KOH) are analytical grade. Deionized water with a resistance of 18 MΩ cm was used in all the experiments. The concentration of both positively charged PEI and negatively charged PAA were diluted to 2.0 g L-1, with pH 11 and 3.0, respectively. The concentration of negatively charged GOx was 0.5 mg/ml. The LPG was firstly treated by H2SO4: H2O2 (7:3) and washed with large amount of deionized water. Next it was dipped into PEI and PAA solutions alternatively, each for 10 min. The process was repeated until 9.5 bilayers of sensing film were deposited. Then it was dipped into GOx solution for 1 hour, and washed with deionized water. III. RESULTS AND DISCUSSION The morphologies of the multilayer films were characterized by atomic force microscopy (AFM). Fig. 1(a)–(c) show the AFM surface images of (PEI/PAA)9, (PEI/PAA)9PEI and (PEI/PAA)9(PEI/GOx)1 multilayer films, respectively. For the (PEI/PAA)9 multilayer film (Fig. 1a), the surface is very rough, and some bumps and holes can be clearly observed. The measured root mean square (RMS) is 22.4 nm. The RMS reduced to be 0.18 nm after the adsorption of PEI layer. And some smaller bumps are distributed on the surface. However, when the negatively charged GOx was deposited on the surface of PEI layer, it covers the smaller bumps, meanwhile, RMS increased to be 6.73 nm. It means that the GOx layer has been successfully deposited on the fiber surface.
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Figure 2 (a) Schematic design of the microfluidic biochip integrated with two fiber-optic sensors. (b) The microscope image of the fabricated microchannels. Figure 1 AFM images for (PEI/PAA)9 (a), (PEI/PAA)9PEI (b) and (PEI/PAA)9(PEI/GOx)1 (c) multilayer films: the left images are the surface morphologies and the right are their 3D presentation.
Figure 2 shows the design of microfluidic biochips in which two fiber-optic sensors have been integrated on the two sidechannels for GO detection. Three inlets have been designed for flowing in of GO and GOx solutions. The two GO inlets can be used to control the injection of GO solutions with different concentrations. For example, a drop of blood can be injected into one inlet, and DI water is injected through another inlet to dilute the sample. Thus, the sample consumption can be greatly reduced, which is very demanding in real clinical applications. Moreover, a microfluidic mixer has been utilized to mix GO solutions homogeneously, which is extremely useful for high-accuracy measurement. SU-8 resist was used to fabricate the mold of microfluidic biochip. After fabrication of the mold, PDMS (Sylgard 184, Dow Corning) was then poured onto the mold and cured at 75 o C for 1 hour. The microfluidic chip was then fabricated after peeling off the patterned PDMS and bonding it upon a glass slide, by the O2 plasma for 5 minutes. Fig. 2(b) displays the fabricated solution mixing part of the microfluidic chip. It can be seen the fabricated mixer is exactly the same with our designed one. And one can also see the surface of the microchannel is very smooth, which makes the solution easily to flow through.
Figure 3 Principle GO biosensor: the GOx layer reacting with GO and the gluconic acid generates, which decreases the pH of solution. As a result, the swelling degree of sensing film increase and the RI decreases following it.
The fabricated fiber-optic GO biosensor was then integrated into the microfluidic chip and sealed to complete the fabrication of biochip. The scheme of sensing mechanism is shown in Fig. 3. As the GOx is deposited on the top layer of PEI/PAA multilayers by electrostatic force, GO will be oxidized by the GOx in the presence of oxygen [3]. During the oxidation process, a new chemical ingredient, gluconic acid, will be generated in GO solutions. Consequently, the pH of GO solution will decrease with the reaction. Since PEI/PAA multilayer film is pH responsive film [4, 7], the swelling degree of PEI/PAA multilayer film increases with the decreasing of pH due to the protonating of PAA and PEI [810]. Therefore, the refractive index of the sensing film will also decrease and eventually be measured by the LPG sensor, which is a highly sensitive fiber-optic refractive index sensor [11].
compared with other reported GO sensors. The biochip has ultra-low detection limitation and small sample consumption [12], and thus has great potential for practical applications. IV. CONCLUSION A microfluidic biochip integrated with fiber-optic biosensor has been demonstrated for GO detection. The fiberoptic sensor was fabricated by LbL self-assembly multilayer sensing film on the surface of LPG. The GO biochip has been tested in the experiments. The results have revealed that the biochip can achieve ultra-low detection limit (1 nM) and fast response, and thus has great potential for e.g. diabetes mellitus detection REFERENCES [1]
Figure 4 (a) Wavelength change of the sensor to different GO concentrations. The inset is the the spectra evolution to GO concentration change. (b) Dynamic response of the biochip.
The biochip was tested in the experiments. Fig 4(a) shows the spectral response of the biochip to GO concentration variation. It can be seen that the wavelength happens red shift with the increment of the concentration of GO. It is due to the oxidation reaction of GO at the existence of catalyst, GOx, which results in a decrease of the pH of GO solution. Therefore, the hydrophilicity of PEI/PAA becomes stronger, which thus increases the swelling degree of PEI/PAA multilayer films. The central wavelength of the LPG sensor stops shifting when the GO concentration exceeds 0.02 mM. It indicates that the pH change induced by the reaction has a saturation behavior. Fig 4(b) shows the measured dynamic response of the biochip to different GO concentrations. One can see that the biochip can resolve tiny change of GO concentration as low as 1 nM. The optical signal becomes stable after one or two tests of the same concentration GO solution, which means the response time of the biochip is around 6 min. It is very fast
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