Emerging ultrafast nucleic acid amplification technologies for next-generation molecular diagnostics.

Over the last decade, nucleic acid amplification tests (NAATs) including polymerase chain reaction (PCR) were an indispensable methodology for diagnosing cancers, viral and bacterial infections owing to their high sensitivity and specificity. Because the NAATs can recognize and discriminate even a few copies of nucleic acid (NA) and species-specific NA sequences, NAATs have become the gold standard in a wide range of applications. However, limitations of NAAT approaches have recently become more apparent by reason of their lengthy run time, large reaction volume, and complex protocol. To meet the current demands of clinicians and biomedical researchers, new NAATs have developed to achieve ultrafast sample-to-answer protocols for the point-of-care testing (POCT). In this review, ultrafast NA-POCT platforms are discussed, outlining their NA amplification principles as well as delineating recent advances in ultrafast NAAT applications. The main focus is to provide an overview of NA-POCT platforms in regard to sample preparation of NA, NA amplification, NA detection process, interpretation of the analysis, and evaluation of the platform design. Increasing importance will be given to innovative, ultrafast amplification methods and tools which incorporate artificial intelligence (AI)-associated data analysis processes and mobile-healthcare networks. The future prospects of NA POCT platforms are promising as they allow absolute quantitation of NA in individuals which is essential to precision medicine.

[1]  Jim F Huggett,et al.  Comparative study of sensitivity, linearity, and resistance to inhibition of digital and nondigital polymerase chain reaction and loop mediated isothermal amplification assays for quantification of human cytomegalovirus. , 2014, Analytical chemistry.

[2]  R. Abramson,et al.  Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[3]  S. Kingsmore,et al.  Comprehensive human genome amplification using multiple displacement amplification , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[4]  Zhenli Qiu,et al.  Near-Infrared-to-Ultraviolet Light-Mediated Photoelectrochemical Aptasensing Platform for Cancer Biomarker Based on Core-Shell NaYF4:Yb,Tm@TiO2 Upconversion Microrods. , 2018, Analytical chemistry.

[5]  Amin Aalipour,et al.  Molecular profiling of single circulating tumor cells from lung cancer patients , 2016, Proceedings of the National Academy of Sciences.

[6]  Vivien Marx,et al.  PCR heads into the field , 2015, Nature Methods.

[7]  R. Snodgrass,et al.  A portable device for nucleic acid quantification powered by sunlight, a flame or electricity , 2018, Nature Biomedical Engineering.

[8]  N. V. Pavlova,et al.  Recent developments in the optimization of thermostable DNA polymerases for efficient applications. , 2004, Trends in biotechnology.

[9]  Takashi Kawana,et al.  Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances. , 2007, Journal of biochemical and biophysical methods.

[10]  Samuel Arvidsson,et al.  QuantPrime – a flexible tool for reliable high-throughput primer design for quantitative PCR , 2008, BMC Bioinformatics.

[11]  Meijin Li,et al.  Bio-bar-code-based photoelectrochemical immunoassay for sensitive detection of prostate-specific antigen using rolling circle amplification and enzymatic biocatalytic precipitation. , 2018, Biosensors & bioelectronics.

[12]  Mehmet Toner,et al.  Magnetic barcode assay for genetic detection of pathogens , 2013, Nature Communications.

[13]  Masih Sherafatian,et al.  Tree-based machine learning algorithms identified minimal set of miRNA biomarkers for breast cancer diagnosis and molecular subtyping. , 2018, Gene.

[14]  Peng Liu,et al.  Integrated DNA purification, PCR, sample cleanup, and capillary electrophoresis microchip for forensic human identification. , 2011, Lab on a chip.

[15]  Ke Li,et al.  Instrument-free point-of-care molecular diagnosis of H1N1 based on microfluidic convective PCR , 2017 .

[16]  Harrison S. Edwards,et al.  The Rotary Zone Thermal Cycler: A Low-Power System Enabling Automated Rapid PCR , 2015, PloS one.

[17]  Daniel S. Chertow,et al.  Next-generation diagnostics with CRISPR , 2018, Science.

[18]  Wasun Chantratita,et al.  Exploring the limits of ultrafast polymerase chain reaction using liquid for thermal heat exchange: A proof of principle. , 2010, Applied physics letters.

[19]  M. Heginbothom,et al.  Evaluation of the Idaho Technology LightCycler PCR for the direct detection of Mycobacterium tuberculosis in respiratory specimens. , 2003, The international journal of tuberculosis and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease.

[20]  Tai Hyun Park,et al.  Mimicking the human smell sensing mechanism with an artificial nose platform. , 2012, Biomaterials.

[21]  Xiongying Ye,et al.  A mechanical cell disruption microfluidic platform based on an on-chip micropump. , 2017, Biomicrofluidics.

[22]  P. Craw,et al.  Isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review. , 2012, Lab on a chip.

[23]  V. Didenko,et al.  DNA probes using fluorescence resonance energy transfer (FRET): designs and applications. , 2001, BioTechniques.

[24]  Dianping Tang,et al.  Photoelectrochemical biosensing of disease marker on p-type Cu-doped Zn0.3Cd0.7S based on RCA and exonuclease III amplification. , 2018, Biosensors & bioelectronics.

[25]  Xiong Ding,et al.  Digital Nucleic Acid Detection Based on Microfluidic Lab-on-a-Chip Devices , 2016 .

[26]  S. Kingsmore,et al.  Multiplexed protein profiling on microarrays by rolling-circle amplification , 2002, Nature Biotechnology.

[27]  N. Thelwell,et al.  Duplex Scorpion primers in SNP analysis and FRET applications. , 2001, Nucleic acids research.

[28]  Taewook Kang,et al.  Bubble-free rapid microfluidic PCR. , 2019, Biosensors & bioelectronics.

[29]  G. Walker,et al.  Strand displacement amplification--an isothermal, in vitro DNA amplification technique. , 1992, Nucleic acids research.

[30]  Derek Tseng,et al.  Inkjet-printed point-of-care immunoassay on a nanoscale polymer brush enables subpicomolar detection of analytes in blood , 2017, Proceedings of the National Academy of Sciences.

[31]  Henry A. Erlich,et al.  Analysis of enzymatically amplified β-globin and HLA-DQα DNA with allele-specific oligonucleotide probes , 1986, Nature.

[32]  James J. Collins,et al.  Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6 , 2018, Science.

[33]  Aydogan Ozcan,et al.  Nucleic acid quantification in the field , 2018, Nature Biomedical Engineering.

[34]  Bong-Hyun Jun,et al.  Graphene Oxide Conjugated Magnetic Beads for RNA Extraction. , 2017, Chemistry, an Asian journal.

[35]  Reinhard Niessner,et al.  DNA-based hybridization chain reaction for amplified bioelectronic signal and ultrasensitive detection of proteins. , 2012, Analytical chemistry.

[36]  Kristen L. Helton,et al.  Microfluidic Overview of Global Health Issues Microfluidic Diagnostic Technologies for Global Public Health , 2006 .

[37]  Daniel Evanko,et al.  Hybridization chain reaction , 2004, Nature Methods.

[38]  Tai Hyun Park,et al.  Fabrication and characterization of a PDMS–glass hybrid continuous-flow PCR chip , 2006 .

[39]  M. Kersaudy-Kerhoas,et al.  Microfluidic blood plasma separation for medical diagnostics: is it worth it? , 2016, Lab on a chip.

[40]  Reinhard Niessner,et al.  Palindromic Molecular Beacon Based Z-Scheme BiOCl-Au-CdS Photoelectrochemical Biodetection. , 2019, Analytical chemistry.

[41]  M. Saad Bhamla,et al.  Hand-powered ultralow-cost paper centrifuge , 2017, Nature Biomedical Engineering.

[42]  Eivind Hovig,et al.  Parallel nanoliter detection of cancer markers using polymer microchips. , 2005, Lab on a chip.

[43]  Bong-Hyun Jun,et al.  Advances in dynamic microphysiological organ-on-a-chip: Design principle and its biomedical application , 2019, Journal of Industrial and Engineering Chemistry.

[44]  Peter Nilsson,et al.  An efficient method to perform milliliter-scale PCR utilizing highly controlled microwave thermocycling. , 2004, Chemical communications.

[45]  Tai Hyun Park,et al.  Nanomaterial-Based Biosensor as an Emerging Tool for Biomedical Applications , 2011, Annals of Biomedical Engineering.

[46]  Zhenli Qiu,et al.  Hybridization chain reaction-based colorimetric aptasensor of adenosine 5'-triphosphate on unmodified gold nanoparticles and two label-free hairpin probes. , 2017, Biosensors & bioelectronics.

[47]  Luke P. Lee,et al.  Toward Integrated Molecular Diagnostic System ($i$ MDx): Principles and Applications , 2014, IEEE Transactions on Biomedical Engineering.

[48]  V. Natarajan,et al.  A power-efficient thermocycler based on induction heating for DNA amplification by polymerase chain reaction , 2004 .

[49]  Rizia Bardhan,et al.  Emerging advances in nanomedicine with engineered gold nanostructures. , 2014, Nanoscale.

[50]  Sanjay Tyagi,et al.  Wavelength-shifting molecular beacons , 2000, Nature Biotechnology.

[51]  Ponnambalam Ravi Selvaganapathy,et al.  A Review on Macroscale and Microscale Cell Lysis Methods , 2017, Micromachines.

[52]  K. Mullis,et al.  Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. , 1988, Science.

[53]  P M Vadgama,et al.  Comparative assessment of chemical and gamma-irradiation procedures for implantable glucose enzyme electrodes. , 2000, Biosensors & bioelectronics.

[54]  Avraham Rasooly,et al.  Rapid DNA amplification using a battery-powered thin-film resistive thermocycler. , 2009, Methods in molecular biology.

[55]  A. Bardelli,et al.  Integrating liquid biopsies into the management of cancer , 2017, Nature Reviews Clinical Oncology.

[56]  V. Venkataraman,et al.  A portable battery-operated chip thermocycler based on induction heating , 2002 .

[57]  C. Schrader,et al.  PCR inhibitors – occurrence, properties and removal , 2012, Journal of applied microbiology.

[58]  Li Jiang,et al.  Solar thermal polymerase chain reaction for smartphone-assisted molecular diagnostics , 2014, Scientific Reports.

[59]  Vincent Q. Vu,et al.  Artificial neural networks in the cancer genomics frontier , 2014 .

[60]  Sanjay Tyagi,et al.  Molecular Beacons: Probes that Fluoresce upon Hybridization , 1996, Nature Biotechnology.

[61]  Gnanavel Venkatesan,et al.  Isothermal Nucleic Acid Amplification System: An Update on Methods and Applications , 2018 .

[62]  A. Eyigor,et al.  Implementation of real‐time PCR to tetrathionate broth enrichment step of Salmonella detection in poultry , 2002, Letters in applied microbiology.

[63]  Brian G. Scrivens,et al.  Strand displacement amplification and homogeneous real-time detection incorporated in a second-generation DNA probe system, BDProbeTecET. , 1999, Clinical chemistry.

[64]  D. Whitcombe,et al.  Detection of PCR products using self-probing amplicons and fluorescence , 1999, Nature Biotechnology.

[65]  Mingqin Chen,et al.  Label-free colorimetric assay for base excision repair enzyme activity based on nicking enzyme assisted signal amplification. , 2014, Biosensors & bioelectronics.

[66]  Dianping Tang,et al.  CdS:Mn quantum dot-functionalized g-C3N4 nanohybrids as signal-generation tags for photoelectrochemical immunoassay of prostate specific antigen coupling DNAzyme concatamer with enzymatic biocatalytic precipitation. , 2017, Biosensors & bioelectronics.

[67]  P. Docker,et al.  Rapid PCR amplification using a microfluidic device with integrated microwave heating and air impingement cooling. , 2010, Lab on a chip.

[68]  Derek Tseng,et al.  Targeted DNA sequencing and in situ mutation analysis using mobile phone microscopy , 2017, Nature Communications.

[69]  Zhenli Qiu,et al.  CdTe/CdSe quantum dot-based fluorescent aptasensor with hemin/G-quadruplex DNzyme for sensitive detection of lysozyme using rolling circle amplification and strand hybridization. , 2017, Biosensors & bioelectronics.

[70]  Wenjuan Yang,et al.  NanoPCR observation: different levels of DNA replication fidelity in nanoparticle-enhanced polymerase chain reactions , 2009, Nanotechnology.

[71]  Chunhai Fan,et al.  Evaluation of gold nanoparticles as the additive in real-time polymerase chain reaction with SYBR Green I dye , 2008, Nanotechnology.

[72]  Victor M Ugaz,et al.  A buoyancy-driven compact thermocycler for rapid PCR. , 2007, Clinics in laboratory medicine.

[73]  Meijin Li,et al.  Reduced graphene oxide/BiFeO3 nanohybrids-based signal-on photoelectrochemical sensing system for prostate-specific antigen detection coupling with magnetic microfluidic device. , 2018, Biosensors & bioelectronics.

[74]  B. van Ginneken,et al.  Deep learning as a tool for increased accuracy and efficiency of histopathological diagnosis , 2016, Scientific Reports.

[75]  K. Mullis,et al.  Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. , 1985, Science.

[76]  M. Ringnér,et al.  Classification and diagnostic prediction of cancers using gene expression profiling and artificial neural networks , 2001, Nature Medicine.

[77]  Vittorio Bianco,et al.  A deep learning-enabled portable imaging flow cytometer for cost-effective, high-throughput, and label-free analysis of natural water samples , 2018, Light: Science & Applications.

[78]  Yasuyoshi Mori,et al.  Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects , 2015, Journal of Microbiology.

[79]  J. Stehr,et al.  Ultra-fast PCR technologies for point-of-care testing , 2017 .

[80]  A Manz,et al.  Chemical amplification: continuous-flow PCR on a chip. , 1998, Science.

[81]  Zhongbin Luo,et al.  Ultrasensitive and label-free electrochemical aptasensor of kanamycin coupling with hybridization chain reaction and strand-displacement amplification. , 2018, Analytica chimica acta.

[82]  D. Issadore,et al.  Combining Machine Learning and Nanofluidic Technology To Diagnose Pancreatic Cancer Using Exosomes. , 2017, ACS nano.

[83]  Elaine Lyon,et al.  Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. , 2004, Clinical chemistry.

[84]  Pratanu Roy,et al.  A Review of Flow and Heat Transfer in Rotating Microchannels , 2013 .

[85]  Michael G. Roper,et al.  Quantitative polymerase chain reaction using infrared heating on a microfluidic chip. , 2012, Analytical chemistry.

[86]  Andrew G. Kirk,et al.  Real time plasmonic qPCR: how fast is ultra-fast? 30 cycles in 54 seconds. , 2017, The Analyst.

[87]  T. Kunkel,et al.  Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase. , 1988, Biochemistry.

[88]  Reza Ghaeini,et al.  A Deep Learning Approach for Cancer Detection and Relevant Gene Identification , 2017, PSB.

[89]  Lyndon Gommersall,et al.  Basic principles of real-time quantitative PCR , 2005, Expert review of molecular diagnostics.

[90]  Yibo Zhang,et al.  Wide-field computational imaging of pathology slides using lens-free on-chip microscopy , 2014, Science Translational Medicine.

[91]  Sai Bi,et al.  Hybridization chain reaction: a versatile molecular tool for biosensing, bioimaging, and biomedicine. , 2017, Chemical Society reviews.

[92]  Victor M. Ugaz,et al.  Novel Convective Flow Based Approaches for High-Throughput PCR Thermocycling , 2004 .

[93]  Daniel Malamud,et al.  Saliva as a diagnostic fluid. , 1993, Dental clinics of North America.

[94]  Mona M. Hella,et al.  DNA Amplification by PCR using Low cost, Programmable Microwave Heating , 2008 .

[95]  Myeong Geun Cha,et al.  Multilayer Ag-Embedded Silica Nanostructure as a Surface-Enhanced Raman Scattering-Based Chemical Sensor with Dual-Function Internal Standards. , 2018, ACS applied materials & interfaces.

[96]  T. Notomi,et al.  Loop-mediated isothermal amplification of DNA. , 2000, Nucleic acids research.

[97]  Zhenli Qiu,et al.  NaYF4:Yb,Er Upconversion Nanotransducer with in Situ Fabrication of Ag2S for Near-Infrared Light Responsive Photoelectrochemical Biosensor. , 2018, Analytical chemistry.

[98]  Musa M Mhlanga,et al.  Using tRNA-linked molecular beacons to image cytoplasmic mRNAs in live cells , 2006, Nature Protocols.

[99]  Stefan Böhringer,et al.  A novel real-time PCR assay for quantitative analysis of methylated alleles (QAMA): analysis of the retinoblastoma locus. , 2004, Nucleic acids research.

[100]  Mauricio D. Coen,et al.  Lab-on-a-Drone: Toward Pinpoint Deployment of Smartphone-Enabled Nucleic Acid-Based Diagnostics for Mobile Health Care , 2016, Analytical chemistry.

[101]  P. Neužil,et al.  Ultra fast miniaturized real-time PCR: 40 cycles in less than six minutes , 2006, Nucleic acids research.

[102]  H Tanke,et al.  Simultaneous A8344G heteroplasmy and mitochondrial DNA copy number quantification in myoclonus epilepsy and ragged-red fibers (MERRF) syndrome by a multiplex molecular beacon based real-time fluorescence PCR. , 2001, Nucleic acids research.

[103]  B. Frey,et al.  Predicting the sequence specificities of DNA- and RNA-binding proteins by deep learning , 2015, Nature Biotechnology.

[104]  Da Xing,et al.  A sample-to-answer, real-time convective polymerase chain reaction system for point-of-care diagnostics. , 2017, Biosensors & bioelectronics.

[105]  Jeong-Woo Choi,et al.  Electrochemical cell lysis device for DNA extraction. , 2010, Lab on a chip.

[106]  Derek Tseng,et al.  Fluorescent imaging of single nanoparticles and viruses on a smart phone. , 2013, ACS nano.

[107]  Jose L Garcia-Cordero,et al.  An Affordable and Portable Thermocycler for Real-Time PCR Made of 3D-Printed Parts and Off-the-Shelf Electronics. , 2018, Analytical chemistry.

[108]  Jana Lauzon,et al.  An inexpensive and portable microchip-based platform for integrated RT-PCR and capillary electrophoresis. , 2008, The Analyst.

[109]  James P Landers,et al.  On-chip pressure injection for integration of infrared-mediated DNA amplification with electrophoretic separation. , 2006, Lab on a chip.

[110]  Jennifer A. Doudna,et al.  CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity , 2018, Science.

[111]  Kiminobu Sugaya,et al.  Handheld battery-operated sample preparation device for qPCR nucleic acid detections using simple contactless pouring , 2018 .

[112]  Dayong Wang,et al.  Deep Learning for Identifying Metastatic Breast Cancer , 2016, ArXiv.

[113]  Sanjay Tyagi,et al.  Multiplex detection of four pathogenic retroviruses using molecular beacons. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[114]  James Clements,et al.  Foldscope: Origami-Based Paper Microscope , 2014, PloS one.

[115]  D. Kuhlmeier,et al.  Isothermal Amplification and Quantification of Nucleic Acids and its Usein Microsystems , 2015 .

[116]  Yassin A. Hassan,et al.  Rapid PCR Thermocycling using Microscale Thermal Convection , 2011, Journal of visualized experiments : JoVE.

[117]  Vincent Miralles,et al.  A Review of Heating and Temperature Control in Microfluidic Systems: Techniques and Applications , 2013, Diagnostics.

[118]  K. Pitkänen,et al.  Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase--an extremely heat stable enzyme with proofreading activity. , 1991, Nucleic acids research.

[119]  Hayden C. Metsky,et al.  Field-deployable viral diagnostics using CRISPR-Cas13 , 2018, Science.

[120]  R. Moreno-Sánchez,et al.  Multi‐biomarker pattern for tumor identification and prognosis , 2011, Journal of cellular biochemistry.

[121]  Bozhi Tian,et al.  Plasmonic Photothermal Gold Bipyramid Nanoreactors for Ultrafast Real-Time Bioassays. , 2017, Journal of the American Chemical Society.

[122]  J P Landers,et al.  Infrared-mediated thermocycling for ultrafast polymerase chain reaction amplification of DNA. , 1998, Analytical chemistry.

[123]  Jane Ru Choi,et al.  Advances in digital polymerase chain reaction (dPCR) and its emerging biomedical applications. , 2017, Biosensors & bioelectronics.

[124]  M. Charreyre,et al.  Plasmonic bipyramids for fluorescence enhancement and protection against photobleaching. , 2014, Nanoscale.

[125]  Zhongbin Luo,et al.  Platinum Nanozyme-Catalyzed Gas Generation for Pressure-Based Bioassay Using Polyaniline Nanowires-Functionalized Graphene Oxide Framework. , 2018, Analytical chemistry.

[126]  Carl T Wittwer,et al.  Extreme PCR: efficient and specific DNA amplification in 15-60 seconds. , 2015, Clinical chemistry.

[127]  Seung-Min Park,et al.  Towards clinically translatable in vivo nanodiagnostics. , 2017, Nature reviews. Materials.

[128]  Yasuyoshi Mori,et al.  Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products , 2008, Nature Protocols.

[129]  Victor M Ugaz,et al.  A pocket-sized convective PCR thermocycler. , 2007, Angewandte Chemie.

[130]  Yu-Cheng Lin,et al.  Enhancing the efficiency of a PCR using gold nanoparticles , 2005, Nucleic acids research.

[131]  Luke P. Lee,et al.  Ultrafast photonic PCR , 2015, Light: Science & Applications.

[132]  Daniel A. Fletcher,et al.  Point-of-care quantification of blood-borne filarial parasites with a mobile phone microscope , 2015, Science Translational Medicine.

[133]  Hasan Kurt,et al.  Employment of nanomaterials in polymerase chain reaction: insight into the impacts and putative operating mechanisms of nano-additives in PCR , 2014 .

[134]  Feng Li,et al.  Amplified detection of T4 polynucleotide kinase activity by the coupled λ exonuclease cleavage reaction and catalytic assembly of bimolecular beacons. , 2014, Analytical chemistry.

[135]  Lide Gu,et al.  Research Progress on Rolling Circle Amplification (RCA)-Based Biomedical Sensing , 2018, Pharmaceuticals.

[136]  Steven Bradshaw,et al.  Microwave heating principles and the application to the regeneration of granular activated carbon , 1998 .

[137]  D. Erickson,et al.  Joule heating and heat transfer in poly(dimethylsiloxane) microfluidic systems. , 2003, Lab on a chip.

[138]  Luke P. Lee,et al.  Self-powered integrated microfluidic point-of-care low-cost enabling (SIMPLE) chip , 2017, Science Advances.

[139]  Daniel J Marchiarullo,et al.  Low-power microwave-mediated heating for microchip-based PCR. , 2013, Lab on a chip.

[140]  Robert M. Dirks,et al.  Triggered amplification by hybridization chain reaction. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[141]  Fred Russell Kramer,et al.  Multicolor molecular beacons for allele discrimination , 1998, Nature Biotechnology.

[142]  Roland Zengerle,et al.  IR thermocycler for centrifugal microfluidic platform with direct on-disk wireless temperature measurement system , 2011 .

[143]  P. Lizardi,et al.  Mutation detection and single-molecule counting using isothermal rolling-circle amplification , 1998, Nature Genetics.

[144]  Bryan Lincoln,et al.  Integrated microfluidic tmRNA purification and real-time NASBA device for molecular diagnostics. , 2008, Lab on a chip.

[145]  Tai Hyun Park,et al.  Microheater based on magnetic nanoparticle embedded PDMS , 2010, Nanotechnology.

[146]  Fred Russell Kramer,et al.  tRNA-linked molecular beacons for imaging mRNAs in the cytoplasm of living cells , 2005, Nucleic acids research.

[147]  Luke P. Lee,et al.  Microphysiological Analysis Platform of Pancreatic Islet β‐Cell Spheroids , 2018, Advanced healthcare materials.

[148]  Xing Chen,et al.  Wirelessly addressable heater array for centrifugal microfluidics and escherichia coli sterilization , 2013, 2013 35th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC).

[149]  Yi Shi,et al.  DeepGene: an advanced cancer type classifier based on deep learning and somatic point mutations , 2016, BMC Bioinformatics.

[150]  J. Maurer Rapid detection and limitations of molecular techniques. , 2011, Annual review of food science and technology.

[151]  Hugo Gamboa,et al.  Machine learning for the meta-analyses of microbial pathogens’ volatile signatures , 2018, Scientific Reports.

[152]  Giorgia Antonelli,et al.  Saliva specimen: a new laboratory tool for diagnostic and basic investigation. , 2007, Clinica chimica acta; international journal of clinical chemistry.

[153]  Peter J. Asiello,et al.  Miniaturized isothermal nucleic acid amplification, a review. , 2011, Lab on a chip.

[154]  Carolyn R. Steffen,et al.  Inhibition mechanisms of hemoglobin, immunoglobulin G, and whole blood in digital and real-time PCR , 2018, Analytical and Bioanalytical Chemistry.

[155]  J. Koehler,et al.  Evaluation of Inhibitor-Resistant Real-Time PCR Methods for Diagnostics in Clinical and Environmental Samples , 2013, PloS one.

[156]  M. J. Broadhurst,et al.  Performance of the GeneXpert Ebola Assay for Diagnosis of Ebola Virus Disease in Sierra Leone: A Field Evaluation Study , 2016, PLoS medicine.

[157]  C. Wittwer,et al.  Automated polymerase chain reaction in capillary tubes with hot air. , 1989, Nucleic acids research.

[158]  Zhongbin Luo,et al.  Photoelectrochemical bioanalysis of antibiotics on rGO-Bi2WO6-Au based on branched hybridization chain reaction. , 2019, Biosensors & bioelectronics.

[159]  J. Compton,et al.  Nucleic acid sequence-based amplification , 1991, Nature.

[160]  Masato Saito,et al.  On-chip quantitative detection of pathogen genes by autonomous microfluidic PCR platform. , 2015, Biosensors & bioelectronics.

[161]  Dar-Bin Shieh,et al.  Handheld energy-efficient magneto-optical real-time quantitative PCR device for target DNA enrichment and quantification , 2016 .