The Study Based On Infra-red LED Mediated PCR Platform Combined With Multi-walled Carbon Nanotubes As Heat Transfer Medium

Point-of-Need(PoN)testing,a new concept which has been proposed to enrich Point-of-Care(PoC)testing,is defined as any diagnostic test that can be performed out of reference laboratories,such as home,patient's bed,ICUs,physician's office or production line,etc.PoC testing relates to human testing whereas PoN testing refers to a much larger scope,including human testing,veterinary testing,industrial testing,argo-food testing,environmental testing,forensics and so on.Since the first applications of PoC testing in 1990 s,microfluidic technology has been used to solve technical problems and it has become part of the diagnostics revolution in the healthcare industry.The emerging field of microfluidic technology in combination with Micro-Electro-Mechanical Systems(MEMS)can provide exciting solutions to PoN testing.Bio-analytical systems based on microfluidics and MEMS can allow for miniaturization and integration of processes onto a chip with benefits including test speed,cost,portability and automation.With the increasing need of monitoring the epidemiology of serious infectious diseases,food hygiene,food additives and pesticide residues,particularly in developing countries with poor infrastructure or on-site detection with the need of rapid results,it is urgent to develop portable,easyto-use,inexpensive and rapid molecular diagnostic tools.Miniaturized PCR instruments utilize both MEMS and microfluidic technology to fulfill the conventional PCR functions onto a portable device that can be used in any condition and capable of rapid target gene identification.Many miniaturized PCR devices have exhibited superior performance than bench-top PCR thermo cyclers in the aspects of cost,ramping speed,portability,integration level,efficiency,etc.Over the years,various on-chip heaters have been demonstrated to improve the speed of temperature cycling.In general,the heaters have two categories,namely contact type(thin films,metal heating blocks and Peltier units)and noncontact type(IR,microwave and laser heater units).The ramping speed of micro-fabricated thin film can achieve 175 °C/s for heating speed and-125 °C/s for cooling speed and fulfill 40 thermal cycles within 6 min.But the contact heaters have the demerit of complexity and cost of fabrication.On the other hand,the compact heater design which reduces the thermal mass of the whole chip is essential to achieve fast temperature cycling speed.The non-contact heaters are more favorable for simple design and flexible system integration.As the heaters do not have to contact the sample chamber,it offers more opportunities to utilize multiple chip designs upon the same platform.Several researchers have successfully integrated IR-mediated or laser assisted heater units to directly heat the PCR sample and achieved amplicon detection.The previous IR-mediated PCR utilizes a high-power tungsten lamp and convex lens to heat the sample directly.This method suffers a great heat loss due to the large thermo mass and has a limitation to conduct multi-chamber PCR at the same time.This paper explores the feasibility of using multi-walled carbon nanotubes as heat transfer medium combined with infrared LED as the heating method in portable PCR instrument.Three sets of PCR platforms,compatibale microfluidic chips and PCR products detection modules have been developed.In these three platforms,the main principle is to control the infrared LED voltage signal through PID algorithm and the pulse width modulation technology.By adjust the output power of the infrared LED,the the heating up speed can be regulated.The cooling control and the PCR product detection methods are different among these PCR platforms.In IR mediated Conducting Oil and CarbOn Nanotube circUlaTing PCR(IR-COCONUT PCR)platform,we designed multi-layer microfluidic chip.A copper layer was used to conduct heat between the PCR chamber layer and the conducting oil layer.The cooling down process was achieved by controlling the voltage signal of a peristaltic pump through PID algorithm and PWM technology.Thus,the heated oil in conducting oil layer will be replaced by the unheated oil in the oil tank.In this part of the experiment,HPV 16 was used as a model analyte to verify the performance of the IRCOCONUT PCR platform.The result shows that the ramping rate of the introduced platform is 1.5 °C/s for heating speed and-2.0 °C/s for cooling speed.This platform fulfills 30 thermal cycles within 50 min which is a match to the conventional bench-top PCR thermo cyclers.The PCR products were verified by lateral flow stripes developed to be compatible with the PCR platform within 25 min.In the second infrared PCR platform,we used 3D printing to design the microfluidic chip mold with micro-wells at the bottom of main channel.The chip can partition sample into bottom wells only by one sample loading step and it will ensure no cross contamination among the partitions.Unlike the microfluidic chips in IR-COCONUT PCR platform,the conducting oil in the microfluidic chip is in contact with the samples in the bottom wells.In this PCR platform,we design the optical components holder for fluorescence detection.The infrared LED is fixed on top of this holder to heat up the conducting oil and carbon nanotubes.The blue LED,band-pass filters and CCD camera which were used for the fluorescence detection were fixed on the lower part of this holder.The cooling down process was achieved by controlling the voltage signal of a USB fan through PID algorithm and PWM technology.The Fluent simulation of sample digitization and heat transfer were conducted by Ansys to optimize the geometry of the bottom-wells and the design of the microfluidic chip.The ramping rate of the PCR platform is 2.5 °C/s for heating and-0.9 °C/s for cooling.PCR fluorescent signal was captured at the end of each cycle.This platform can successfully detect three types of HPV DNA and the results are correspondence with the hospital tests.Then we developed the IR MEdiated RNA Isothermal RT-PCR(IRMERIT PCR)platform.This IR-MERIT PCR platform consists three modules: temperature control module,fluorescence detection module and positioning module.Since this platform conducts isothermal amplification,the hardware for cooling down control can be elimated.We utilized 3D printing to fabricate the major components in positiong module which can detect the fluorescence signals in different chambers flexibly.We also designed the microfluidic chip with 3D micro channels by utilizing patterend materials.This method has shown a great potential to integrate micro pumps,micro mixers or micro valves into the microfluidic chip in a simple way.Then we utilize this IR-MERIT PCR platform,mirrofluidic chip and photodiode to detect the Ureaplasma urealyticum(UU),Chlamydia trachomatis(CT)and Mycoplasma genitourae(MG)infection simutanously.The design of these three PCR platforms and their compatible microfluidic chips have shown a great potential for rapid diagnostic of gene or infectious diseases in filed or developing countries.It also offers a good solution for accurate and portable point-of-need PCR device.