Experimental investigation of confinement effect in single molecule amplification via real-time digital PCR on a multivolume droplet array SlipChip.

BACKGROUND: Digital polymerase chain reaction (digital PCR) is an important quantitative nucleic acid analysis method in both life science research and clinical diagnostics. One important hypothesis is that by physically constraining a single nucleic acid molecule in a small volume, the relative concentration can be increased therefore further improving the analysis performance, and this is commonly defined as the confinement effect in digital PCR. However, experimental investigation of this confinement effect can be challenging since it requires a microfluidic device that can generate partitions of different volumes and an instrument that can monitor the kinetics of amplification. (96). RESULTS: Here, we developed a real-time digital PCR system with a multivolume droplet array SlipChip (Muda-SlipChip) that can generate droplet of 125 nL, 25 nL, 5 nL, and 1 nL by a simple "load-slip" operation. In the digital region, by reducing the volume, the relative concentration is increased, the amplification kinetic can be accelerated, and the time to reach the fluorescence threshold, or Cq value, can be reduced. When the copy number per well is much higher than one, the relative concentration is independent of the partition volume, thus the amplification kinetics are similar in different volume partitions. This system is not limited to studying the kinetics of digital nucleic acid amplification, it can also extend the dynamic range of the digital nucleic acid analysis by additional three orders of magnitude by combining a digital and an analog quantification algorithm. (140). SIGNIFICANCE: In this study, we experimentally investigated for the first time the confinement effect in the community of digital PCR via a new real-time digital PCR system with a multivolume droplet array SlipChip (Muda-SlipChip). And a wider dynamic range of quantification methods compared to conventional digital PCR was validated by this system. This system provides emerging opportunities for life science research and clinical diagnostics. (63).

Multiplex Digital Immunoassays with Reduced Pre-partition Reaction via Massively Parallel Reagent Delivery on a Bead-Based SlipChip.

The emergence of digital immunoassays has advanced the sensitivity of protein analysis to ultrahigh sensitivity at the attomolar level. However, the background signal generated by the premixing of immunocomplexes and fluorogenic substrates can limit the precise quantification, especially in multiplexed assays. Herein, a bead-based SlipChip (bb-SlipChip) microfluidic device capable of massively parallel two-step sample loading is presented. The background signal can be suppressed through a two-step loading mechanism. Specifically, encapsulate the beads into the microwells first and then, through a slipping process, deliver the fluorogenic substrate in parallel into 281,200 microwells of 68 fL to perform the digital immunoassay. The quantification capability is demonstrated with a duplex assay of IL-6 and IL-10, achieving a limit of detection of 5.2 and 15.3 fg/mL, which is approximately 2-3 times improved compared to a commercial Simoa system. The bb-SlipChip provides a robust and universal method for digital immunoassay and can be extended to higher multiplexed detection as well as other biomedical applications involving microbeads.

Multiplex Digital Polymerase Chain Reaction on a Droplet Array SlipChip for Analysis of KRAS Mutations in Pancreatic Cancer.

Pancreatic cancer is a terminal disease with high mortality and very poor prognosis. A sensitive and quantitative analysis of KRAS mutations in pancreatic cancer provides a tool not only to understand the biological mechanisms of pancreatic cancer but also for diagnosis and treatment monitoring. Digital polymerase chain reaction (PCR) is a promising tool for KRAS mutation analysis, but current methods generally require a complex microfluidic handling system, which can be challenging to implement in routine research and point-of-care clinical diagnostics. Here, we present a droplet-array SlipChip (da-SlipChip) for the multiplex quantification of KRAS G12D, V, R, and C mutant genes with the wild-type (WT) gene background by dual color (FAM/ROX) fluorescence detection. This da-SlipChip is a high-density microwell array of 21,696 wells of 200 pL in 4 by 5424 microwell format with simple loading and slipping operation. It does not require the same precise alignment of microfeatures on the different plates that are acquired by the traditional digital PCR SlipChip. This device can provide accurate quantification of both mutant genes and the WT KRAS gene. We collected tumor tissue, paired normal pancreatic tissue, and other normal tissues from 18 pancreatic cancer patients and analyzed the mutation profiles of KRAS G12D, V, R, and C in these samples; the results from the multiplex digital PCR on da-SlipChip agree well with those of next-generation sequencing (NGS). This da-SlipChip moves digital PCR closer to the practical point-of-care applications not only for detecting KRAS mutations in pancreatic cancer but also for other applications that require precise nucleic acid quantification with high sensitivity.

Parallel multistep digital analysis SlipChip demonstrated with the quantification of nucleic acid by digital LAMP-CRISPR.

Digital biological analysis compartmentalizes targets of interest, such as nucleic acids, proteins, and cells, to a single event level and performs detection and further investigation. Microfluidic-based digital biological analysis methods, including digital PCR, digital protein analysis, and digital cell analysis, have demonstrated superior advantages in research applications and clinical diagnostics. However, most of the methods are still based on a one-step "divide and detect" strategy, and it is challenging for these methods to perform further parallel manipulation of reaction partitions to achieve "divide, manipulate, and analyze" capabilities. Here, we present a parallel multistep digital analysis (PAMDA) SlipChip for the parallel multistep manipulation of a large number of droplets for digital biological analysis, demonstrated by the quantification of SARS-CoV-2 nucleic acids by a two-step digital isothermal amplification combined with clustered regularly interspaced short palindromic repeats (CRISPR). This PAMDA SlipChip utilizes a "chain-of-pearl" channel with a self-partitioning droplet formation mechanism that does not require the precise alignment of microfeatures for fluidic loading as the traditional SlipChip design. This device can first generate 2400 3.2 nanoliter droplets to perform digital loop-mediated isothermal amplification (LAMP) and then deliver reagents containing Cas12a protein and crRNA to each individual partition in parallel to simultaneously initiate digital CRISPR detection by a simple multistep slipping operation. This PAMDA SlipChip not only provides a promising tool to perform digital CRISPR with a flexible assay and workflow design but can also be applied for a broad range of applications in digital biological analysis that require multistep manipulation of partitions in parallel.

Slip formation of a high-density droplet array for nucleic acid quantification by digital LAMP with a random-access system.

Digital nucleic acid analysis (digital NAA) is an important tool for the precise quantification of nucleic acids. Various microfluidic-based approaches for digital NAA have been developed, but most methods require complex auxiliary control instruments, cumbersome device fabrication, or inconvenient preparation processes. A SlipChip is a microfluidic device that can generate and manipulate liquid partitions through simple movements of two microfluidic plates in close contact. However, the traditional SlipChip requires accurate alignment of microfeatures on different plates; therefore, the dimensions of the microwells and density of partitions can be constrained. Here, we developed a droplet array SlipChip (da-SlipChip) that can form droplets of various sizes at high density in a single slipping step. This process does not require precise overlapping microfeatures on different plates; therefore, the design flexibility and partition density can be significantly increased. We quantified SARS-CoV-2 nucleic acids extracted from the COVID-19 pseudovirus by digital loop-mediated isothermal amplification (LAMP) on a da-SlipChip with 21 696 of 0.25 nL droplets, and the results were in good agreement with those of the commercial digital PCR method of Stilla. Furthermore, we demonstrated a random-access system with a single-throughput fluorescence imager and a stackable thermal control instrument with nine independent heating modules. This random-access system with the da-SlipChip can greatly improve the total throughput and flexibility for digital isothermal nucleic acid quantification and significantly reduce the total waiting time.

Self-partitioning SlipChip for slip-induced droplet formation and human papillomavirus viral load quantification with digital LAMP.

Human papillomavirus (HPV) is one of the most common sexually transmitted infections worldwide, and persistent HPV infection can cause warts and even cancer. Nucleic acid analysis of HPV viral DNA can be very informative for the diagnosis and monitoring of HPV. Digital nucleic acid analysis, such as digital PCR and digital isothermal amplification, can provide sensitive detection and precise quantification of target nucleic acids, and its utility has been demonstrated in many biological research and medical diagnostic applications. A variety of methods have been developed for the generation of a large number of individual reaction partitions, a key requirement for digital nucleic acid analysis. However, an easily assembled and operated device for robust droplet formation without preprocessing devices, auxiliary instrumentation or control systems is still highly desired. In this paper, we present a self-partitioning SlipChip (sp-SlipChip) microfluidic device for the slip-induced generation of droplets to perform digital loop-mediated isothermal amplification (LAMP) for the detection and quantification of HPV DNA. In contrast to traditional SlipChip methods, which require the precise alignment of microfeatures, this sp-SlipChip utilized a design of "chain-of-pearls" continuous microfluidic channel that is independent of the overlapping of microfeatures on different plates to establish the fluidic path for reagent loading. Initiated by a simple slipping step, the aqueous solution can robustly self-partition into individual droplets by capillary pressure-driven flow. This advantage makes the sp-SlipChip very appealing for the point-of-care quantitative analysis of viral load. As a proof of concept, we performed digital LAMP on a sp-SlipChip to quantify human papillomaviruses (HPVs) 16 and 18 and tested this method with fifteen anonymous clinical samples.

Slip Molding for Precision Fabrication of Microparts.

Microparts with precise sizes, custom shapes, and a wide selection of materials have various applications, including biomedical microelectromechanical systems (MEMS), drug delivery, single-cell studies, and tissue engineering. Janus microparts containing multiple components are also demonstrated for biomolecule analysis, cell-cell interaction studies, and self-assembly. Small-footprint, affordable, and rapid technologies to fabricate microparts with customized morphologies and a wide selection of materials are highly desired. This paper reports on a SlipChip-based microfluidic molding method to control the interface for the synthesis of microparts-on-demand (mPods) with fast and easy loading-slipping-solidification operations that do not require pumps, masks, or other auxiliary fluidic control instruments. This method is based on the relative movement of two microfluidic plates that are in close contact, and the size and shape of the microparts can be accurately controlled by the geometry of the microcavities imprinted on the contacting surfaces of these microfluidic plates. To demonstrate the capability of this method, mPods of different sizes and various shapes are presented with photosensitive resin via a photopolymerization reaction. The synthesis of two-layer Janus microparts is also demonstrated by a slip overmolding method. This SlipChip-based molding method can offer new opportunities for producing customized microparts with great flexibility for a broad spectrum of applications.

Slip-driven microfluidic devices for nucleic acid analysis.

Slip-driven microfluidic devices can manipulate fluid by the relative movement of microfluidic plates that are in close contact. Since the demonstration of the first SlipChip device, many slip-driven microfluidic devices with different form factors have been developed, including SlipPAD, SlipDisc, sliding stripe, and volumetric bar chart chip. Slip-driven microfluidic devices can be fabricated from glass, quartz, polydimethylsiloxane, paper, and plastic with various fabrication methods: etching, casting, wax printing, laser cutting, micromilling, injection molding, etc. The slipping operation of the devices can be performed manually, by a micrometer with a base station, or autonomously, by a clockwork mechanism. A variety of readout methods other than fluorescence microscopy have been demonstrated, including both fluorescence detection and colorimetric detection by mobile phones, direct visual detection, and real-time fluorescence imaging. This review will focus on slip-driven microfluidic devices for nucleic acid analysis, including multiplex nucleic acid detection, digital nucleic acid quantification, real-time nucleic acid amplification, and sample-in-answer-out nucleic acid analysis. Slip-driven microfluidic devices present promising approaches for both life science research and clinical molecular diagnostics.