In this work, we present a simple and equipment-free system for discretizing samples into tens of thousands of discrete volumes in tens of seconds. Unlike conventional sample discretization systems that...
This
paper proposes a simple and robust method for spontaneously
digitizing aqueous samples into a high-density microwell array. The
method is based on an oil-triggered template-confined dewetting phenomenon.
To realize the dewetting-induced sample digitization, an aqueous sample
is first infused into a networked microwell array (NMA) through a
pre-degassing-based self-pumping mechanism, and an immiscible oil
phase is then applied over the surface of NMA chip to induce the templated
dewetting. Due to periodic interfacial tension heterogeneity, such
dewetting ruptures the sample at the thinnest parts (i.e., connection channels) and spontaneously splits the sample into
droplets in individual microwells. Without requiring any complex pumping
or valving systems, this method can discretize a sample into tens
of thousands of addressable droplets in a matter of minutes with nearly
98% usage. To demonstrate the utility and universality of this self-digitization
method, we exploited it to discretize samples into 40 233 wells
for a digital PCR assay, the digital quantification of bacteria, the
self-assembly of spherical colloidal photonic crystals, and the spherical
crystallization of drugs. We believe this facile technique will provide
a substantial benefit to many compartmentalized assays or syntheses
where it is necessary to partition samples into a large number of
small individual volumes.
Digital polymerase chain reaction (dPCR) is emerging
as a powerful
method for nucleic acid detection due to its unprecedented sensitivity
and precision. However, most current dPCR platforms are inherently
limited by their low multiplexing ability due to primer-pair cross
interactions and spectral overlap of available fluorophores. Here,
we present a novel and robust method for multiplexing dPCR that is
free from primer dimerization and fluorescence channel number limitation,
enabling highly precise and multiplexed detection of nucleic acid
targets. By prestoring target-specific primers and probes in different
storage chambers, the method physically separates reactions and thus
avoids the primer-pair cross interactions and spectral overlap of
different fluorescent probes that usually occur within a single-tube
reaction. Furthermore, a dissolvable delay valve (DDV) is embedded
between each pair of the reagent prestorage chamber and reaction microwell
array. Such a DDV configuration allows full reconstitution of the
prestored reagents and then generates a uniform concentration distribution
of the reconstituted reagents across the entire reaction microwell
array, which is favorable for achieving reliable and robust multiplex
dPCR assays. We demonstrated the feasibility of this method by performing
an eight-plex dPCR assay targeting the seven most common point mutations
in Kirsten rat sarcoma viral oncogene homologue (KRAS) and a reference
sequence (wild-type KRAS allele).
Digital bioassays are powerful methods to detect rare analytes from complex mixtures and study the temporal processes of individual entities within biological systems. In digital bioassays, a crucial first step is the discretization of samples into a large number of identical independent partitions. Here, we developed a rapid and facile sample partitioning method for versatile digital bioassays. This method is based on a detachable self-digitization (DSD) chip which couples a reversible assembly configuration and a predegassing-based self-pumping mechanism to achieve an easy, fast and large-scale sample partitioning. The DSD chip consists of a channel layer used for loading sample and a microwell layer used for holding the sample partitions. Benefitting from its detachability, the chip avoids a lengthy oil flushing process used to remove the excess sample in loading channels and can compartmentalize a sample into more than 100,000 wells of picoliter volume with densities up to 14,000 wells/cm2 in less than 30 s. We also demonstrated the utility of the proposed method by applying it to digital PCR and digital microbial assays.
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