Smartphone-Imaged HIV-1 Reverse-Transcription Loop-Mediated Isothermal Ampliflcation(RT-LAMP) on a Chip from Whole Blood

Viral load measurements are an essential tool for the long-term clinical care of human immunodeficiency virus （HIV）-positive individuals. The gold standards in viral load instrumentation, however, are still too limited by their size, cost, and sophisticated operation for these measurements to be ubiquitous in remote settings with poor healthcare infrastructure, including parts of the world that are disproportionately affected by HIV infection. The challenge of developing a point-of-care platform capable of making viral load more accessible has been frequently approached but no solution has yet emerged that meets the practical requirements of low cost, portability, and ease-of-use. In this paper, we perform reverse-transcription loop-mediated isothermal amplification （RT-LAMP） on minimally processed HIV-spiked whole blood samples with a microfluidic and silicon microchip platform, and perform fluorescence measurements with a consumer smartphone. Our integrated assay shows amplification from as few as three viruses in a - 60 nL RT- LAMP droplet, corresponding to a whole blood concentration of 670 viruses per μL of whole blood. The technology contains greater power in a digital RT-LAMP approach that could be scaled up for the determination of viral load from a finger prick of blood in the clinical care of HIV-positive individuals. We demonstrate that all aspects of this viral load approach, from a drop of blood to imaging the RT-LAMP reaction, are compatible with lab-on-a-chip components and mobile instrumentation.

Label-free SARS-CoV-2 detection and classification using phase imaging with computational specificity

Efforts to mitigate the COVID-19 crisis revealed that fast,accurate,and scalable testing is crucial for curbing the current impact and that of future pandemics.We propose an optical method for directly imaging unlabeled viral particles and using deep learning for detection and classification.An ultrasensitive interferometric method was used to image four virus types with nanoscale optical path-length sensitivity.Pairing these data with fluorescence images for ground truth,we trained semantic segmentation models based on U-Net,a particular type of convolutional neural network.The trained network was applied to classify the viruses from the interferometric images only,containing simultaneously SARS-CoV-2,H1N1(influenza-A virus),HAdV(adenovirus),and ZIKV(Zika virus).Remarkably,due to the nanoscale sensitivity in the input data,the neural network was able to identify SARS-CoV-2 vs.the other viruses with 96%accuracy.The inference time for each image is 60 ms,on a common graphic-processing unit.This approach of directly imaging unlabeled viral particles may provide an extremely fast test,of less than a minute per patient.As the imaging instrument operates on regular glass slides,we envision this method as potentially testing on patient breath condensates.The necessary high throughput can be achieved by translating concepts from digital pathology,where a microscope can scan hundreds of slides automatically.

A 3D-printed platform for modular neuromuscular motor units

A complex and functional living cellular system requires the interaction of one or more cell types to perform specific tasks,such as sensing,processing,or force production.Modular and flexible platforms for fabrication of such multi-cellular modules and their characterization have been lacking.Here,we present a modular cellular system,made up of multi-layered tissue rings containing integrated skeletal muscle and motor neurons(MNs)embedded in an extracellular matrix.The MNs were differentiated from mouse embryonic stem cells through the formation of embryoid bodies(EBs),which are spherical aggregations of cells grown in a suspension culture.The EBs were integrated into a tissue ring with skeletal muscle,which was differentiated in parallel,to create a co-culture amenable to both cell types.The multi-layered rings were then sequentially placed on a stationary three-dimensionalprinted hydrogel structure resembling an anatomical muscle–tendon–bone organization.We demonstrate that the site-specific innervation of a group of muscle fibers in the multi-layered tissue rings allows for muscle contraction via chemical stimulation of MNs with glutamate,a major excitatory neurotransmitter in the mammalian nervous system,with the frequency of contraction increasing with glutamate concentration.The addition of tubocurarine chloride(a nicotinic receptor antagonist)halted the contractions,indicating that muscle contraction was MN induced.With a bio-fabricated system permitting controllable mechanical and geometric attributes in a range of length scales,our novel engineered cellular system can be utilized for easier integration of other modular“building blocks”in living cellular and biological machines.