Zitian Chen

Digital Polymerase Chain Reaction in an Array of Femtoliter Polydimethylsiloxane Microreactors

We developed a simple, compact microfluidic device to perform high dynamic-range digital polymerase chain reaction (dPCR) in an array of isolated 36-femtoliter microreactors. The density of the microreactors exceeded 20000/mm(2). This device, made from polydimethylsiloxane (PDMS), allows the samples to be loaded into all microreactors simultaneously. The microreactors are completely sealed through the deformation of a PDMS membrane. The small volume of the microreactors ensures a compact device with high reaction efficiency and low reagent and sample consumption. Future potential applications of this platform include multicolor dPCR and massively parallel dPCR for next generation sequencing library preparation.

Selection of DNA-Encoded Small Molecule Libraries Against Unmodified and Non-Immobilized Protein Targets**

The selection of DNA-encoded libraries against biological targets has become an important discovery method in chemical biology and drug discovery, but the requirement of modified and immobilized targets remains a significant disadvantage. With a terminal protection strategy and ligand-induced photo-crosslinking, we show that iterated selections of DNA-encoded libraries can be realized with unmodified and non-immobilized protein targets.

Spinning micropipette liquid emulsion generator for single cell whole genome amplification

Many on-chip approaches that use flow-focusing to pinch the continuous aqueous phase into droplets have become the most popular methods that provide monodisperse emulsion droplets. However, not every lab can easily adapt a microfluidic workflow into their familiar protocols. We develop an off-chip approach, spinning micro-pipette liquid emulsion (SiMPLE) generator, to produce highly stable monodisperse water-in-oil emulsions using a moving micropipette to disperse the aqueous phase in an oil-filled microcentrifuge tube. This method provides a simple way to produce picoliter-size droplets in situ with no dead volume during emulsification. With SiMPLE, single-cell emulsion whole genome amplification was performed to demonstrate that this novel method can seamlessly be integrated with experimental operations and supplies that most researchers are familiar with. The SiMPLE generator has effectively lowered the technical difficulties in applications relying on emulsion droplets.

Highly accurate fluorogenic DNA sequencing with information theory–based error correction

Eliminating errors in next-generation DNA sequencing has proved challenging. Here we present error-correction code (ECC) sequencing, a method to greatly improve sequencing accuracy by combining fluorogenic sequencing-by-synthesis (SBS) with an information theory–based error-correction algorithm. ECC embeds redundancy in sequencing reads by creating three orthogonal degenerate sequences, generated by alternate dual-base reactions. This is similar to encoding and decoding strategies that have proved effective in detecting and correcting errors in information communication and storage. We show that, when combined with a fluorogenic SBS chemistry with raw accuracy of 98.1%, ECC sequencing provides single-end, error-free sequences up to 200 bp. ECC approaches should enable accurate identification of extremely rare genomic variations in various applications in biology and medicine.

A high-throughput imaging system to quantitatively analyze the growth dynamics of plant seedlings

Most current methods for analyzing the growth rate of plant seedlings are limited to low-throughput experimental configurations. We have developed an automatic system to investigate the dynamics of the growth of hypocotyls using Arabidopsis as model. This system is able to capture time-lapse infrared images of 24 seedlings automatically, with a spatial resolution of 2 μm per pixel and temporal interval of 5 min. Seedling length is rapidly calculated using automated geometric image-processing algorithms. With this high-throughput platform, we have investigated the genotype dependent difference of growth patterns, as well as the response to plant hormone - ethylene. Our analyses suggest that cytoskeleton function is not required in ethylene-induced hypocotyl inhibition. This novel integrative method can be applied to large-scale dynamic screening of plants, as well as any other image-based biological studies related to dynamic growth.

Fluorogenic Sequencing Using Halogen‐Fluorescein‐Labeled Nucleotides

Fluorogenic sequencing is a sequencing‐by‐synthesis technology that combines the advantages of pyrosequencing and fluorescence detection. With native duplex DNA as the major product, we employ polymerase to incorporate the complement‐ arily matched terminal phosphate‐labeled fluorogenic nucleotides into the DNA template and release halogen‐fluorescein as the reporter. This red‐emitting fluorophore successfully avoids spectral overlap with the autofluorescence background of the flow chip. We fully characterized the enzymatic reaction kinetics of the new substrates, and performed a 35‐base sequencing experiment with 60 reaction cycles. Our achievement expands the substrate repertoire for fluorogenic sequencing, and extends the spectral range to obtain better signal‐to‐background performance.