SPEED:an integrated,smartphone-operated,handheld digital PCR Device for point-of-care testing

This study elaborates on the design,fabrication,and data analysis details of SPEED,a recently proposed smartphonebased digital polymerase chain reaction(dPCR)device.The dPCR chips incorporate partition diameters ranging from 50μm to 5μm,and these partitions are organized into six distinct blocks to facilitate image processing.Due to the superior thermal conductivity of Si and its potential for mass production,the dPCR chips were fabricated on a Si substrate.A temperature control system based on a high-power density Peltier element and a preheating/cooling PCR protocol user interface shortening the thermal cycle time.The optical design employs four 470 nm light-emitting diodes as light sources,with filters and mirrors effectively managing the light emitted during PCR.An algorithm is utilized for image processing and illumination nonuniformity correction including conversion to a monochromatic format,partition identification,skew correction,and the generation of an image correction mask.We validated the device using a range of deoxyribonucleic acid targets,demonstrating its potential applicability across multiple fields.Therefore,we provide guidance and verification of the design and testing of the recently proposed SPEED device.

An integrated microfluidic platform for nucleic acid testing

This study presents a rapid and versatile low-cost sample-to-answer system for SARS-CoV-2 diagnostics.The system integrates the extraction and purification of nucleic acids,followed by amplification via either reverse transcriptionquantitative polymerase chain reaction(RT–qPCR)or reverse transcription loop-mediated isothermal amplification(RT–LAMP).By meeting diverse diagnostic and reagent needs,the platform yields testing results that closely align with those of commercial RT-LAMP and RT‒qPCR systems.Notable advantages of our system include its speed and costeffectiveness.The assay is completed within 28 min,including sample loading(5 min),ribonucleic acid(RNA)extraction(3 min),and RT-LAMP(20 min).The cost of each assay is≈$9.5,and this pricing is competitive against that of Food and Drug Administration(FDA)-approved commercial alternatives.Although some RNA loss during on-chip extraction is observed,the platform maintains a potential limit of detection lower than 297 copies.Portability makes the system particularly useful in environments where centralized laboratories are either unavailable or inconveniently located.Another key feature is the platform’s versatility,allowing users to choose between RT‒qPCR or RT‒LAMP tests based on specific requirements.

Synthetic Gene Circuit-Based Assay with Multilevel Switch Enables Background-Free and Absolute Quantification of Circulating Tumor DNA

Circulating tumor DNA(ctDNA)detection has found widespread applications in tumor diagnostics and treatment,where the key is to obtain accurate quantification of ctDNA.However,this remains challenging due to the issue of background noise associated with existing assays.In this work,we developed a synthetic gene circuit-based assay with multilevel switch(termed CATCH)for background-free and absolute quantification of ctDNA.The multilevel switch combining a small transcription activating RNA and a toehold switch was designed to simultaneously regulate transcription and translation processes in gene circuit to eliminate background noise.Moreover,such a multilevel switch-based gene circuit was integrated with a Cas9 nickase H840A(Cas9n)recognizer and a molecular beacon reporter to form CATCH for ctDNA detection.The CATCH can be implemented in one-pot reaction at 35°C with virtually no background noise,and achieve robust absolute quantification of ctDNA when integrated with a digital chip(i.e.,digital CATCH).Finally,we validated the clinical capability of CATCH by detecting drug-resistant ctDNA mutations from the plasma of 76 non-small cell lung cancer(NSCLC)patients,showing satisfying clinical sensitivity and specificity.We envision that the simple and robust CATCH would be a powerful tool for next-generation ctDNA detection.