Study On DNA Immobilization On Metal Microdots In Microfluidic Channel

Microfluidics is expected to have many advantages such as greatly reducing reagent consumption, shortening reaction time, reducing costs, and have high potential integration , therefore it is becoming one of important driving forces in the development of a new generation of bio-analytical techniques. It is currently an important development direction to carry out the integration of microfluidic principles and technical means with microarray technology including gene chip. In order to achieve the integration of DNA microarray technology with microfluidic approach, the firstly important step is that DNA probes are required to be immobilized into microchannel, which is the very basic component in microfluidics.The fabrication of microchannels is realized usually by use of the traditional photolithography and thereafter the emerging soft lithography. Therefore, the sealing step is a must in the state-of-the-art integration of microarray with microfluidic techniques, however, which brings about many adverse consequences in subsequent biological analysis.In this paper, we carried out the fabrication of microchannel by use of a newly developed method named microwire-molding based on the strategy of soft lithography. Metal microdots and its array in a microchannel could be formed when two pieces of microwires were placed in a way of crossing-contacted each other in the process of microwire-molding. The limitations occurred in the sealing step required by the conventional technique could therefore be overcome. Starting from this point, this article focused on the subsequent issues that how to realize the immobilization of DNA on the microdots located on the inner wall of the microchannel, and how to perform the hybridization experiment so as to gain the viewpoint of its feasibility.Three types of DNA immobilization experiments in the microchannel have been carried out: thiol-DNA covalently bonding with gold microdot, electric adsorbing of DNA on stainless steel microdots, co-immobilization of DNA and polypyrrole on stainless steel microdots. And for the later two cases, the hybridization experiments have been performed.The results showed that: (1) A visible fluorescent microdot could be seen within the microchannel only after 5 hours when the thiol-DNA-FAM covalently bonding with the gold microdot, suggesting significant shortening the time needed in such covalent binding than conventional. (2) With negative charge of DNA had been adsorbed on the cathode, but not on the anode at DC as our expectation, the intensity of fluorescence microdot has been influenced with DNA concentration, electrolyte concentration, microdot size, the distance between electrodes and electric conduction time.①I n the micro-channel of 20μm diameter, no visible fluorescence microdot formed in the end. The crossing dot area of Stainless steel wire which diameters were 40, 60, 80 and 100μm was directly proportional to the adsorbed DNA fluorescence intensity.②C onstant conditions in other cases, the DNA concentration increased gradually in the time between 0.00305-12.5nmol/ml, fluorescence intensity continued to increase with the time going on, but when DNA concentration was more than 0.78125nmol/ml the impaction of it is small. When the DNA concentration was 0.00305nmol/ml because the DNA concentration was small enough, it is difficult in a relatively short time to form a visible DNA fluorescence microdot.③Other conditions in the case of a constant concentration, when NaCL concentration is between the 0.063-0.25mol / L and increased gradually, the adsorbed ssDNA microdot fluorescence intensity continued to increase, but the impact became small when it is more than 0.25mol / L When the concentration of NaCL in 0.063mol/L, it is difficult to form the effective DNA fluorescence microdot on the stainless steel microdot in the 50s.④when interelectrode distance increased, the adsorbed ssDNA microdot fluorescence intensity continued to decrease, it is difficult to form the effective DNA fluorescence microdot on the stainless steel microdot when the interelectrodewas more 520μm.⑤when electric conduction was between the 0-200s ,and increased gradually, the adsorbed ssDNA microdot fluorescence intensity continued to increase, in the sixth seconds and controllable, that is, a visible fluorescent microdot has been formed, but the impact became small when it is more than 100s. (3)after coating a layer of PDMS on the stainless steel microdots surface, polypyrrole has been modified on the surface of microdot through the electrochemical method, and at the same time target DNA has been immobilized too. After that, the current change experiments about the temperature and hybridization had been finished, the results appear that in the optimum temperature conditions, the current signal increased 32.03 times, while at room temperature increased only 3.51-fold; after hybridization, the current signal increased 127.273 times completely than the basis current.; without hybridization behavior, the current signal increase of only 21.4 times bigger.In summary, The three ways of immobilization of DNA probes on the metal microdot electrodes in microchannel were successfully performed, and the feasibility of the later two was confirmed with their hybridization to the complementary DNA probes. The results suggest an integration route of DNA immobilization with label-less detection of hybridization, and also suggest a rudiment step to the integration of microarray with microfluidics.