Study Of Bacterium Biofilms Based On The Microfluidic Chip Integrated With Photoelectricity Sensors

Bacteria often exist in the form of bacterial biofilms at the living environment,which has brought many adverse effects to public health,medical treatment,and environment.In recent years,researchers at home and abroad have paid much attention to the research field including the development of efficient detection methods of bacterial biofilms,exploring the relationship between the formation of bacterial biofilms and quorum sensing(QS),and looking for effective strategies to inhibit bacterial biofilms.In this work,several optoelectronic sensing chips integrated with electrochemistry,fluorescence,surface enhanced Raman spectroscopy(SERS)detection technologies were designed and prepared for the non-destructive monitoring of bacterial biofilms,and the tracking of quorum sensing in bacterial biofilms.By using the fabricated optoelectronic sensing chips,the formation cycle of biofilms and the change of QS signal molecules were comprehensively studied.The effects of traditional antibiotics,active components of traditional Chinese Medicine,and nano anti-bacterial materials on bacterial biofilms formation were further explored.The main research work and achievements are as follows:Part?:In situ fluorescence observation of Tannin-Ag NPs inhibiting E.coli biofilms on microfluidic chip.A multi-channel microcavity array PDMS-glass chip was designed and fabricated for the observation of bacterial biofilms by fluorescence.Tannic acid modified silver nanoparticles(Tannin-Ag NPs)were synthesized with uniform particle size about42.37nm.The minimum inhibitory concentration(MIC)of Tannin-Ag NPs against E.coli was as low as 16?g/ml.Based on the expression of green fluorescent protein in E.coli,the inhibitory effect of Tannin-Ag NPs on the formation of E.coli biofilms was investigated by real-time fluorescence observation and detection on the chip.The results showed Tannin-Ag NPs at 1/2MIC concentration strongly inhibited the formation of E.coli biofilms in the chip,and its inhibitory effect was stronger than the same concentration of tannic acid and Ag NPs.Tannin-Ag NPs also changed the inherent formation period of E.coli biofilms,and shortened the growth period of E.coli biofilms from 15 h to 9 h.Part?:In situ and nondestructive monitoring of biofilms formation of E.coli and Salmonella using electrical impedance chip.An electrical impedance sensor chip integrated with interdigital microelectrodes was designed and fabricated,and the formation periodic of E.coli and Salmonella biofilms was distinguished by the impedance analysis method.Interdigital gold microelectrodes were integrated with the bottom of the chip to monitor the growth of bacterial biofilms in the frequency range of 1?100 k Hz at a disturbance voltage of 100m V for 48 h.The impedance spectra of E.coli and Salmonella biofilms were further analyzed by using the equivalent circuit analysis model including parameters of biofilms capacitance C_b and biofilms resistance R_b.The impedance fitting results showed that the impedance signals of E.coli and Salmonella biofilms were different,and the change of the time nodes of C_b and R_b between the two bacterial biofilms were also different,indicating that the formation process of E.coli and Salmonella biofilms could be distinguished by impedance method.Finally,the analysis results of the formation process of bacterial biofilms based on the impedance sensor chip were verified by crystal violet staining method.Part?:Real-time monitoring of electroactive QS signal molecules during biofilms formation of Pseudomonas aeruginosa by an electrochemical sensor chip.An electrochemical sensor chip integrated with microelectrodes array was designed and fabricated for in-situ and real-time monitoring of pyocyanin during biofilms formation of Pseudomonas aeruginosa.Microelectrodes composed of working electrode,reference electrode and counter electrode were integrated into the chip.A layer of uniform gold nanoparticles with particle size of about 100 nm was prepared on the surface of the working electrode by electrochemical deposition method.The detection limit of pyocyanin was as low as 100 n M by differential pulse voltammetry(DPV).There was a good linear relationship between the concentration of pyocyanin and the peak current in the concentration range of 100 n M?200?M,and the correlation coefficient was 0.9997.The dynamic change of pyocyanin during the biofilms formation of Pseudomonas aeruginosa was monitored by DPV on the chip for 72 h.The results showed that the concentration of pyocyanin was lower than 1?M in the early adhesion stage of Pseudomonas aeruginosa biofilms;the concentration of pyocyanin increased rapidly in the growth stage of Pseudomonas aeruginosa biofilms and the concentration of pyocyanin increased to more than 100?M;the concentration of pyocyanin showed a downward trend in the spreading period of Pseudomonas aeruginosa biofilms.At the same time,the effect of curcumin on Pseudomonas aeruginosa biofilms was studied.It was found that curcumin at lower than MIC could significantly reduce the production level of pyocyanin and the maximum concentration was reduced to 38.4?M.Part?:Detection and analysis of the interaction between different anti-microbial agents and Pseudomonas aeruginosa biofilms by SERS chip.A SERS chip integrated were designed and fabricated for the monitoring of the interaction process between different anti-microbial agents and Pseudomonas aeruginosa biofilms.By using chemical self-assembly technology,a homogeneous and stable nano-silver SERS substrate was integrated into the chip.The apparent enhancement factor of nano-silver SERS substrate was 1.84×10⁸,which greatly enhanced the Raman signal of Pseudomonas aeruginosa biofilms.The detection limit of QS signal molecule pyocyanin was as low as 10^-9mol/L on the SERS substrate.The interaction effects of ceftazidime,curcumin and tannin on Pseudomonas aeruginosa biofilms were also monitored for 72 hours by the SERS chip.SERS spectra showed that the intensity of Raman peak at 650 cm^-1 and 728 cm^-1 increased within 0?12h,indicating that the biofilms of Pseudomonas aeruginosa gradually grew.Druing the period of 24?36 h,the intensity of Raman peak at 1350 cm^-1 was high,which suggeted that pyocyanin molecule was produced.The intensity of Raman peak at 1350 cm^-1decreased and the concentration of pyocyanin started to decrease druing 36?72 h.The inhibition effect of ceftazidime,curcumin and tannin on the growth of Pseudomonas aeruginosa biofilms and the production of pyocyanin was ceftazidime>curcumin>tannin.Furthermore,the monitoring results of SERS chip were verified by mass spectrometry.The results showed that the analytical method based on SERS chip could be used to study the bacterial biofilms and its interaction with anti-microbial drugs.