Light-Addressable Potentiometric Sensor (LAPS) Combined With Microfluidics And Its Applications In Cells And Organoids Detection

Cell metabolism is one of the most basic biological mechanisms of life.The study of cell physiology and functions is of great significance.Cell metabolism is related to a variety of ions in the extracellular environment.For example,the energy metabolism generates the energy and releases the acidic waste products,leading to p H change in the extracellular environment.Na⁺and K⁺are actively transported through the Na⁺/K⁺-ATPase to maintain the cell osmotic pressure and resting potential;Ca²⁺is one of secondary messengers to transmit intracellular signals.Therefore,cell metabolism can be reflected by detecting the above ions.As a member of the ion sensors,light-addressable potentiometric sensor(LAPS)plays an important role in the field of biochemical detection due to its high sensitivity and light addressability.LAPS is a kind of field-effect-based semiconductor biochemical sensor,which combines light and electronics.The light addressability makes it possible to define arbitrary shapes and sizes of measured areas at particular positions on the sensor surface.LAPS has been widely used in biochemical detection because of the advantages like simple structure,high sensitivity,and easy combination with microfluidics.In this dissertation,a variety of functional LAPS sensor units and detection systems are established based on sensor fabrication,detection unit package,microfluidics production,sensing material preparation,optical system and electric circuit design,used for cell acidification detection,bacteria glycolysis detection,medium ions detection and organoid impedance image detection.The functional application of LAPS sensor system in the biochemical detection field is developed.The main content and innovations of this dissertation are given as follows:1.A LAPS detection system integrated with microfluidics is proposed,eliminates the complex structure of the traditional microphysiometer,and elementarily realizes the function of real-time extracellular acidification(ECAR)detectionIn this work,a polydimethylsiloxane(PDMS)microcavity is combined with LAPS to construct a simple,sensitive,non-invasive microfluidic detection system for real-time detection of ECAR.The functions of traditional microphysiometer are elementarily realized by using a simple and miniaturized sensor unit,and the optical observation of cells in the microchamber is not affected.The medium and drugs can be automatically delivered to the cell chamber,and the required sample volume is within tens of microliters.The PDMS microcavity can be used for cell culture,and the cells can be directly observed through an optical microscope.The bubble trap avoids the interference of bubbles in the flow path.Hep G2 human hepatoma cells is used for the ECAR detection,and extra glucose and anticancer drug doxorubicin are used to assess the effect on cell metabolism.The problem that the detection unit of conventional microphysiometer is too complicated to perform optical observation is solved by the proposed sensor system,which provides a new platform for cell metabolism detection and drug efficacy evaluation.2.A microfluidic LAPS detection system containing a porous membrane is designed and established,overcomes the difficulty in detecting non-adherent bacteria in a fluidic environment,and elementarily realizes the real-time detection of sugar metabolism of lactic acid bacteria(LAB)The biochemical detection function of LAPS is developed from cell to bacteria.This is the first time to detect the non-adherent bacteria with a microfluidic LAPS system.Based on the previous work,a microchamber for bacterial detection is constructed using the Transwell devices,in which the polycarbonate microporous membrane can protect the bacterial liquid from being washed away,while allowing the solution exchange in the chamber.An O-ring is used to maintain the height of the detection chamber,while limiting the bacteria to the effective detection area.The partially thinned sensor chip is fabricated to achieve the backside illumination,which eliminates the light interference causing by the turbid bacterial liquid.The characteristic of the sensor and the solution exchange capacity of the porous membrane microchamber are verified.Artificial saliva is used to simulate the oral environment where LAB is located.Lactobacillus rhamnosus is used for sugar metabolism detection,and different concentrations of glucose and sugar substitutes are used to verify the effect on the metabolism of LAB.The sensor system overcomes the difficulty in detecting non-adherent targets in a fluidic environment and provides a elementarily microfluidic detection platform that can be used for bacterial metabolism detection.3.A design method of multiplexed ion-sensitive LAPS(ISLAPS)detection system based on silicone-rubber membrane is proposed,elementarily achieves the simultaneous detection of Ca²⁺,Na⁺,K⁺and H⁺in the extracellular environmentThe biochemical detection function of LAPS is developed from single ion detection to multi-ion detection.LAPS is combined with a variety of silicone-rubber ion-sensitive membranes(ISMs)for the first time to establish a program-controlled multiplexed detection system.The simultaneous detection of the four ions is realized with single-channel instrument hardware.And the detection targets can be expanded by only adding other sensing membranes due to the light addressability of LAPS.A conducting polymer internal layer is pre-modified to the sensor surface to inhibit water layer formation,and the spin-coating method is applied to ensure the uniformity of the modified membrane.With the aid of data acquisition device and translation stage,the modulated light illuminates the detection sites in sequence,and the response of the four ions can be obtained within 1 minute.After the characteristic test of the ion-sensitive membranes,the function of the multiplexed ISLAPS detection system is verified by a variety of spiked real medium samples,and the detection performance is evaluated compared with other similar studies.The problem of single detection target in traditional ion sensors is solved by the multiplexed ISLAPS and provides a multi-parameter ion detection platform.4.A design of high-resolution laser scanning imaging system based on the ultra-thin silicon LAPS is proposed,provides the supplementary impedance information for traditional optical images,and acquires the impedance images of organoidsThe biochemical detection function of LAPS is developed to the organoid level,expanding the one-dimensional concentration information to two-dimensional image information,which makes full use of the light addressability of LAPS.A novel LAPS with ultra-thin silicon layer on glass substrate(silicon on glass,SOG)is designed and fabricated.Data acquisition device and self-made circuit module are used to replace bulk instruments like the potentiostat and lock-in amplifier;an optical system is built to focus the light spot below 400?m;a new scanning path is adopted to obtain a quick acquisition of high-resolution image;a self-written Lab VIEW software realizes the real-time display of LAPS scanning images.The image resolution of LAPS is determined by the image of PDMS pattern.Olfactory epithelium organoids of mouse are used for scanning imaging experiments,and Triton X-100 is selected to verify the SOG-LAPS system function of the organoid impedance image acquisition.The sensor detection system can provide supplementary impedance information elementarily for traditional optical methods and provides a new platform for organoid detection and drug efficacy evaluation.