Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection System for Viral DNA Sensing

Abstract

A fully Integrated Micropillar Polydimethylsiloxane Accurate CRISPR deTection (IMPACT) system is developed for viral DNA detection. This powerful system is patterned with high-aspect-ratio micropillars to enhance reporter probe binding. After surface modification and probe immobilization, the CRISPR-Cas12a/crRNA complex is injected into the fully enclosed microchannel. With the presence of a double-stranded DNA target, the CRISPR enzyme is activated and denatures the single-stranded DNA reporters from the micropillars. This collateral cleavage releases fluorescence reporters into the assay, and the intensity is linearly proportional to the target DNA concentration ranging from 0.1 to 10 nM. Importantly, this system does not rely on the traditional dye-quencher-labeled probe, thus reducing the fluorescence background presented in the assay. Furthermore, our one-step detection protocol is performed on-chip at isothermal conditions (37 °C) without using complicated and time-consuming off-chip probe hybridization and denaturation. This miniaturized and fully packed IMPACT chip demonstrates sensitive and accurate DNA detection within 120 min and paves ways to the next-generation point-of-care diagnostics, responding to emerging and deadly pathogen outbreaks.

Figure 1

(a) Schematic of the clustered regularly interspaced short palindromic repeats (CRISPR)-based IMPACT chip DNA detection. (b) Photograph of the IMPACT chip. The dashed white box indicates regions patterned with micropillars. (c) SEM image of the micropillars. Inset: high magnification image. (d) Static water contact angle measurement on both flat and micropillar surfaces before (orange) and after (green) surface treatment. (e) Schematic of the surface treatment protocol, ssDNA probe binding, and CRISPR detection. (f) ASFV target DNA sequence and the corresponding crRNA sequence. (g) Fluorescent image of the channel which received chemical treatment and streptavidin binding (green fluorescence). (h) Fluorescent ssDNA-covered IMPACT chip, showing red fluorescence.

Figure 2

Washing data for flat and micropillar channels. The treated flat and microchannel samples were coated with streptavidin. (a) Integrated intensity of supernatant after first wash with 75 μL of DI water through channel. (b) Comparison of integrated intensity of the supernatant after washing 75 μL of DI water through the channel the first and second time. Inset shows the uncorrected emission curve of washed reporter probe for the treated micropillar channel. (c) Integrated intensity of reporter probes released with UV light exposure. Error bars are standard deviation of the mean.

Figure 3

Released reporter probe intensity versus incubation time (10 min to 24 h). Treated samples received surface modification and streptavidin treatment before photocleavable reporter probe incubation (1 nmol). The sample was retrieved via UV photocleavage. Error bars are standard deviation of the mean.

Figure 4

Released ssDNA reporter probe integrated intensity for flat and micropillar channels with a DNA input load of 1, 0.1, and 0.01 nmol. UV photocleavage was used to retrieve the ssDNA reporter probes. The skewness of each dataset is presented by boxplot.

Figure 5

IMPACT chip performance with different ASFV target DNA concentrations. (a) Uncorrected emission curve of the collected supernatant with a target concentration ranging from 0.1 to 10 nM. The shaded area indicates the range used for integration to obtain data for part (b) (550–600 nm). (b) Integrated intensity of the collected supernatant versus target concentration on a logarithmic scale (Pearson’s R = 0.9778). Error bars are standard deviation of the mean. Parameter “a” in the equation indicates the expected intensity value (on a log scale) for a sample with 1 nM of target DNA present. Parameter b represents the slope of the curve fit on the log–log scale, meaning for every order of magnitude increase in concentration, the expected logarithmic intensity will increase by 0.6821.