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

Mengchao Yu,Rustem F Ismagilov,Hua Wang,Xiaoying Chen,Feng Shen,Haijun Qu,Liang Ma,Lei Xu,Min Li,Weiyuan Lv

doi:10.1021/acs.analchem.9b01270

Abstract

Serial dilution is a commonly used technique that generates a low-concentration working sample from a high-concentration 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.

Highlights

Serial dilution is a common laboratory practice used to generate solutions with relatively low concentrations from high-concentration samples
It is widely used to set up an array, such as arrays in 96- or 384-well plate format, for screening conditions or performing quantification over a large dynamic range.[1−4] In practice, serial dilution is usually performed with tedious multiple manual pipetting steps or an often expensive robotic liquid handling system
The top and bottom plates were assembled to an initial position: the mother microwells on the top plate and their complementary parts on the bottom plate partially overlapped to establish a Type-I continuous fluidic path, which consists of mother microwells and complementary mother wells; the fluidic ducts on the top plate and the daughter microwells on the bottom plate partially overlapped to form Type-II continuous fluidic paths, which consist of daughter microwells (Figure 1B)

Summary

■ RESULTS AND DISCUSSION

The sd-SlipChip consists of two opposing microfluidic plates that have microfabricated microwells and fluidic ducts on the contact surface. We designed an eight-step sd-SlipChip with a fewer than 600 total wells to perform digital LAMP quantification over a large dynamic range of 7 orders of magnitude (Figure S2) This sd-SlipChip contains 8 rows of daughter microwells and 64 of 12 nL microwells per row for daughter microwells. By preserving the high concentration sample stock in the 64 of 12 nL complementary mother microwells (top row in Figure 3G) and utilizing all 8 rows of daughter microwells, the full dynamic range from the 9 rows can be expanded beyond the total dilution ratio. For the 12 HBV-positive samples, the quantification results from serial dilution digital LAMP on the sd-SlipChip were in good agreement with the results from standard HBV clinical protocols (Figure 3J). Further statistical tools or computational algorithms that perform additional analysis with data can provide more comprehensive results and additional insight on the error rate for the sd-SlipChip, but these approaches are beyond the scope of this paper

■ CONCLUSION

■ ACKNOWLEDGMENTS

■ REFERENCES