Multistep SlipChip for the Generation of Serial Dilution Nanoliter Arrays and Hepatitis B Viral Load Quantification by Digital Loop Mediated Isothermal Amplification

Abstract

Serial dilution is a commonly used technique that generates a low-concentration working sample from a highconcentration stock solution and is used to set up screening conditions over a large dynamic range for biological study, optimization of reaction conditions, drug screening, etc. Creating an array of serial dilutions usually requires cumbersome manual pipetting steps or a robotic liquid handling system. Moreover, it is very challenging to set up an array of serial dilutions in nanoliter volumes in miniaturized assays. Here, a multistep SlipChip microfluidic device is presented for generating serial dilution nanoliter arrays in high throughput with a series of simple sliding motions. The dilution ratio can be precisely predetermined by the volumes of mother microwells and daughter microwells, and this paper demonstrates devices designed to have dilution ratios of 1:1, 1:2, and 1:4. Furthermore, an eight-step serial dilution SlipChip with a dilution ratio of 1:4 is applied for digital loop-mediated isothermal amplification (LAMP) across a large dynamic range and tested for hepatitis B viral load quantification with clinical samples. With 64 wells of each dilution and fewer than 600 wells in total, the serial dilution SlipChip can achieve a theoretical quantification dynamic range of 7 orders of magnitude.

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

“…Next, the glass plate was submerged in a chromium etchant solution described previously. (Yu et al, 2019) The glass etching was conducted at 40 °C in a shaking water bath (frequency: 50 rpm/min). A profilometer (Bruker, Billerica, MA) was used to monitor the etching depth.…”

“…SlipChips are a type of microfluidic device that can partition liquid by the relative movement of two microfluidic plates with imprinted microwells or fluidic ducts on the contacting surface. (Cai et al, 2014;Du et al, 2009;Li et al, 2010;Liu et al, 2010;Shen et al, 2014Shen et al, , 2011bShen et al, , 2010Sun et al, 2013;Yu et al, 2019) They do not require additional auxiliary systems, such as pumps, pneumatic systems, and electronic systems, for control and manipulation of the liquid. However, these traditional SlipChips generally require the precise alignment of microwells or fluidic ducts on different plates to establish or break fluidic paths, which may limit their widespread application in different settings.…”

Slip-driven microfluidic devices for nucleic acid analysis

“…To demonstrate the robustness and easy-to-use nature of a SlipChip device, a six-year-old volunteer performed an experiment to generate thousands of nanoliter microdroplets on an injection-molded plastic SlipChip with simple manual operations. 21 The use of these slip-driven microfluidic devices has been demonstrated in multiplex nucleic acid analysis for the identification of target molecules, [22][23][24] digital nucleic acid quantification of target molecules, [25][26][27][28][29][30][31] real-time nucleic acid analysis, [32][33][34] and samplein-answer-out nucleic acid analysis. 20,23 In addition to nucleic acid analysis, slip-driven microfluidic devices have also been applied to the study of protein crystallization, 35,36 immunoassays, [37][38][39][40] cell and bacterial cultivation, 19,41,42 bacterial chemotaxis, 43 separation by isoelectric focusing 44,45 or electrophoresis, 46 antimicrobial susceptibility testing (AST), 47 sample storage, 48 etc.…”

Microfluidic SlipChip device for multistep multiplexed biochemistry on a nanoliter scale

“…Multistep SlipChip protocols involving serial dilutions have been demonstrated. 28,30 This work adds a capability to perform complete droplet transfer (previously droplets were split by slipping and retained in the wells) in addition to the option to retrieve samples after reaction without cross-contamination. SlipChips have also been demonstrated for multiplexing.…”

“…Importantly, the device contains a set of features into which the user can pre-spot reagents that can be used to uniquely identify the contents of any one well from any other well after they are pooled. So instead of offering on-device readout only as demonstrated previously, 9,28,30 this device is designed for the user to be able to extract the products of on-device reactions for further analysis. We demonstrate the utility of this multistep device to perform multiplexed reactions by performing a seven-step workflow: transcript barcoding for multiplexed cDNA sequencing library preparation for total RNA sequencing (RNAtag-Seq).…”