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A versatile snap chip for high-density sub-nanoliter chip-to-chip reagent transfer.

Li H, Munzar JD, Ng A, Juncker D - Sci Rep (2015)

Bottom Line: Misalignment, which for direct transfer ranged from 150-250 μm, was reduced to <40 μm for double transfer.The versatility of the snap chip is illustrated with a 4-plex homogenous enzyme inhibition assay analyzing 128 conditions with precise timing.The versatility and high density of the snap chip with double transfer allows for the development of high throughput reagent transfer protocols compatible with a variety of applications.

View Article: PubMed Central - PubMed

Affiliation: 1] Biomedical Engineering Department, McGill University, Montréal, QC, H3A 0G1, Canada [2] McGill University and Genome Quebec Innovation Centre, McGill University, Montréal, QC, H3A 0G1, Canada.

ABSTRACT
The coordinated delivery of minute amounts of different reagents is important for microfluidics and microarrays, but is dependent on advanced equipment such as microarrayers. Previously, we developed the snap chip for the direct transfer of reagents, thus realizing fluidic operations by only manipulating microscope slides. However, owing to the misalignment between arrays spotted on different slides, millimeter spacing was needed between spots and the array density was limited. In this work, we have developed a novel double transfer method and have transferred 625 spots cm(-2), corresponding to >10000 spots for a standard microscope slide. A user-friendly snapping system was manufactured to make liquid handling straightforward. Misalignment, which for direct transfer ranged from 150-250 μm, was reduced to <40 μm for double transfer. The snap chip was used to quantify 50 proteins in 16 samples simultaneously, yielding limits of detection in the pg/mL range for 35 proteins. The versatility of the snap chip is illustrated with a 4-plex homogenous enzyme inhibition assay analyzing 128 conditions with precise timing. The versatility and high density of the snap chip with double transfer allows for the development of high throughput reagent transfer protocols compatible with a variety of applications.

No MeSH data available.


Related in: MedlinePlus

Fluorescence microscope image and results of multiplexed enzymatic inhibition assay of thrombin.(a) Fluorescence image obtained after 15 min of on-chip enzymatic activity. Experimental results and one phase exponential fits of inhibition assays conducted using (b) the snap chip or (c) conventional microcentrifuge tube assays; PPACK and Argatroban inhibit thrombin in a dose-dependent manner, while PMSF and Leupeptin do not, as expected (n = 3 independent experiments, error bars are standard deviation). The difference observed for the inhibition curves between the snap chip and microcentrifuge tube assays for PPACK is accounted for when the reagents in the microcentrifuge tube assay were stored at room temperature for 15 minutes prior to carrying out the assay (PPACK-RT).
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f6: Fluorescence microscope image and results of multiplexed enzymatic inhibition assay of thrombin.(a) Fluorescence image obtained after 15 min of on-chip enzymatic activity. Experimental results and one phase exponential fits of inhibition assays conducted using (b) the snap chip or (c) conventional microcentrifuge tube assays; PPACK and Argatroban inhibit thrombin in a dose-dependent manner, while PMSF and Leupeptin do not, as expected (n = 3 independent experiments, error bars are standard deviation). The difference observed for the inhibition curves between the snap chip and microcentrifuge tube assays for PPACK is accounted for when the reagents in the microcentrifuge tube assay were stored at room temperature for 15 minutes prior to carrying out the assay (PPACK-RT).

Mentions: The fluorescence generated from substrate turnover is shown in Fig. 6a, demonstrating the dose-dependent inhibition of thrombin within individual droplets. Note that the fluorescence image of the droplets appears as a torus due to a lensing effect of the hemispherical droplets under the microscope. Data analysis of the resulting fluorescence is shown in a normalized format in Fig. 6b. The results highlight the effective inhibition of human alpha-thrombin by the direct thrombin inhibitors D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK) and Argatroban. Meanwhile, even at a 1000-fold higher concentration, PMSF showed no inhibitory activity against thrombin, with substrate turnover rates equivalent to that of Leupeptin, a non-targeting control for these experiments. Importantly, the results obtained with the snap chip are in good agreement with those obtained with microcentrifuge tubes that use 1000 times more sample, as shown in Fig. 6c. The data obtained with the snap chip and the benchmark, large volume microcentrifuge assays for Argatroban are in excellent agreement, generating the same dose-specific inhibition of thrombin over the range of inhibitor concentrations tested. However, for PPACK, the dose-dependent thrombin inhibition in the snap chip assay indicated decreased inhibitory activity. It is known from the literature that the chloromethyl ketone moiety is hydrolyzed in basic solutions3233, and the assay was carried out at a pH of 7.5, which could lead to significant hydrolysis. The reagents used in the large-scale microcentrifuge tube assay were kept on ice until initiation of the reaction, hence hydrolysis would be expected to be slowed, whereas for the nanoscale snap chip assays, printing and chip handling were carried out at room temperature, potentially leading to a more pronounced inhibitor hydrolysis. To assess this hypothesis, we performed a large volume assay in microcentrifuge tubes with pre-incubation of PPACK and substrate at room temperature (PPACK-RT) for 15 min prior to mixing with thrombin, Fig. 6c. When following the same temperature cycles, the inhibition curves of the snap chip and microcentrifuge assays show the same kinetics. Argatroban is not prone to hydrolysis under these conditions, further corroborating this analysis. In summary, these results illustrate that the snap chip can be used in place of traditional large-volume methods to screen candidate inhibitors against targets of interest in a high throughput format. However, if quantitative results are desired, care must be taken to compensate for secondary effects such as temperature or oxygen sensitivity that may skew the results obtained with the snap chip.


A versatile snap chip for high-density sub-nanoliter chip-to-chip reagent transfer.

Li H, Munzar JD, Ng A, Juncker D - Sci Rep (2015)

Fluorescence microscope image and results of multiplexed enzymatic inhibition assay of thrombin.(a) Fluorescence image obtained after 15 min of on-chip enzymatic activity. Experimental results and one phase exponential fits of inhibition assays conducted using (b) the snap chip or (c) conventional microcentrifuge tube assays; PPACK and Argatroban inhibit thrombin in a dose-dependent manner, while PMSF and Leupeptin do not, as expected (n = 3 independent experiments, error bars are standard deviation). The difference observed for the inhibition curves between the snap chip and microcentrifuge tube assays for PPACK is accounted for when the reagents in the microcentrifuge tube assay were stored at room temperature for 15 minutes prior to carrying out the assay (PPACK-RT).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493572&req=5

f6: Fluorescence microscope image and results of multiplexed enzymatic inhibition assay of thrombin.(a) Fluorescence image obtained after 15 min of on-chip enzymatic activity. Experimental results and one phase exponential fits of inhibition assays conducted using (b) the snap chip or (c) conventional microcentrifuge tube assays; PPACK and Argatroban inhibit thrombin in a dose-dependent manner, while PMSF and Leupeptin do not, as expected (n = 3 independent experiments, error bars are standard deviation). The difference observed for the inhibition curves between the snap chip and microcentrifuge tube assays for PPACK is accounted for when the reagents in the microcentrifuge tube assay were stored at room temperature for 15 minutes prior to carrying out the assay (PPACK-RT).
Mentions: The fluorescence generated from substrate turnover is shown in Fig. 6a, demonstrating the dose-dependent inhibition of thrombin within individual droplets. Note that the fluorescence image of the droplets appears as a torus due to a lensing effect of the hemispherical droplets under the microscope. Data analysis of the resulting fluorescence is shown in a normalized format in Fig. 6b. The results highlight the effective inhibition of human alpha-thrombin by the direct thrombin inhibitors D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK) and Argatroban. Meanwhile, even at a 1000-fold higher concentration, PMSF showed no inhibitory activity against thrombin, with substrate turnover rates equivalent to that of Leupeptin, a non-targeting control for these experiments. Importantly, the results obtained with the snap chip are in good agreement with those obtained with microcentrifuge tubes that use 1000 times more sample, as shown in Fig. 6c. The data obtained with the snap chip and the benchmark, large volume microcentrifuge assays for Argatroban are in excellent agreement, generating the same dose-specific inhibition of thrombin over the range of inhibitor concentrations tested. However, for PPACK, the dose-dependent thrombin inhibition in the snap chip assay indicated decreased inhibitory activity. It is known from the literature that the chloromethyl ketone moiety is hydrolyzed in basic solutions3233, and the assay was carried out at a pH of 7.5, which could lead to significant hydrolysis. The reagents used in the large-scale microcentrifuge tube assay were kept on ice until initiation of the reaction, hence hydrolysis would be expected to be slowed, whereas for the nanoscale snap chip assays, printing and chip handling were carried out at room temperature, potentially leading to a more pronounced inhibitor hydrolysis. To assess this hypothesis, we performed a large volume assay in microcentrifuge tubes with pre-incubation of PPACK and substrate at room temperature (PPACK-RT) for 15 min prior to mixing with thrombin, Fig. 6c. When following the same temperature cycles, the inhibition curves of the snap chip and microcentrifuge assays show the same kinetics. Argatroban is not prone to hydrolysis under these conditions, further corroborating this analysis. In summary, these results illustrate that the snap chip can be used in place of traditional large-volume methods to screen candidate inhibitors against targets of interest in a high throughput format. However, if quantitative results are desired, care must be taken to compensate for secondary effects such as temperature or oxygen sensitivity that may skew the results obtained with the snap chip.

Bottom Line: Misalignment, which for direct transfer ranged from 150-250 μm, was reduced to <40 μm for double transfer.The versatility of the snap chip is illustrated with a 4-plex homogenous enzyme inhibition assay analyzing 128 conditions with precise timing.The versatility and high density of the snap chip with double transfer allows for the development of high throughput reagent transfer protocols compatible with a variety of applications.

View Article: PubMed Central - PubMed

Affiliation: 1] Biomedical Engineering Department, McGill University, Montréal, QC, H3A 0G1, Canada [2] McGill University and Genome Quebec Innovation Centre, McGill University, Montréal, QC, H3A 0G1, Canada.

ABSTRACT
The coordinated delivery of minute amounts of different reagents is important for microfluidics and microarrays, but is dependent on advanced equipment such as microarrayers. Previously, we developed the snap chip for the direct transfer of reagents, thus realizing fluidic operations by only manipulating microscope slides. However, owing to the misalignment between arrays spotted on different slides, millimeter spacing was needed between spots and the array density was limited. In this work, we have developed a novel double transfer method and have transferred 625 spots cm(-2), corresponding to >10000 spots for a standard microscope slide. A user-friendly snapping system was manufactured to make liquid handling straightforward. Misalignment, which for direct transfer ranged from 150-250 μm, was reduced to <40 μm for double transfer. The snap chip was used to quantify 50 proteins in 16 samples simultaneously, yielding limits of detection in the pg/mL range for 35 proteins. The versatility of the snap chip is illustrated with a 4-plex homogenous enzyme inhibition assay analyzing 128 conditions with precise timing. The versatility and high density of the snap chip with double transfer allows for the development of high throughput reagent transfer protocols compatible with a variety of applications.

No MeSH data available.


Related in: MedlinePlus