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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

Fluorescent image of an assay slide and standard binding curves for 50 proteins simultaneously measured using the snap chip.(a) Fluorescent micrograph of a representative assay slide with 16 replicate arrays incubated in serial standard dilutions. (b) Close-up of the array within the dashed box in (a), with 153 spots and a 400 μm center-to-center spacing. Scale bar: 2 mm. (c) Standard curves for 50 proteins (for CA 15-3 the concentration is in unit/mL). Error bars represent standard deviations calculated from three independent experiments. Negative control fluorescence signals generated with 0 ng/mL protein concentrations are shown as separate data points.
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f5: Fluorescent image of an assay slide and standard binding curves for 50 proteins simultaneously measured using the snap chip.(a) Fluorescent micrograph of a representative assay slide with 16 replicate arrays incubated in serial standard dilutions. (b) Close-up of the array within the dashed box in (a), with 153 spots and a 400 μm center-to-center spacing. Scale bar: 2 mm. (c) Standard curves for 50 proteins (for CA 15-3 the concentration is in unit/mL). Error bars represent standard deviations calculated from three independent experiments. Negative control fluorescence signals generated with 0 ng/mL protein concentrations are shown as separate data points.

Mentions: To demonstrate the multiplexing capabilities of the snap chip using the double transfer method, an antibody colocalization microarray targeting breast cancer biomarkers252627, cancer-related proteins, and cytokines28 was performed according to a protocol outlined in Fig. 2. The immunoassay consisted of a total of 50 different antibodies and GFP calibrations spots, spotted in triplicate per pad, resulting in 2448 spots per slide. Aminosilane functionalized slides were used as transfer slides for the cAbs and dAbs. These slides act as good transfer surfaces due to their moderate contact angle, which helps to widen spots and therefore facilitate alignment, while on the other hand resulting in high droplets extending tens of micrometers above the surface, so as to ensure contact with the assay slide. The assay slides were coated with ~12 μm thick nitrocellulose pads, which provided a large binding capacity for cAbs and also facilitated the subsequent transfer of dAb droplets by partially absorbing the liquid. After spotting and transferring the cAb, the assay slide and the transfer slide with dAbs were stored in a −20 °C freezer. To run an assay, the slides were first rehydrated as described in detail in the methods section. Seven serial dilutions of each protein and a negative control (no protein) were incubated in the eight wells of each column of nitrocellulose pads on the assay slide, Fig. 5a. Variations in spot sizes between triplicate spots for antibody pairs, which match across all array pads, can be attributed to volumetric errors in the inkjet spotting, depending on the spotted antibody, Fig. 5b. The variations in spot size did not affect immunoassay data analysis because of the homogeneity across all the pads, and might be mitigated with larger volumes for each spot. The fluorescence intensity was quantified, and standard curves were generated, and are grouped based on the binding ranges of each antibody pair and the signal intensity, Fig. 5c. The LODs of 35 proteins were found to be in the pg/mL range, with the lowest being 3.3 pg/mL for CCL3, SI Table 2. The LOD might be improved by further optimizing the assay and storage conditions, since the current ACM format allows the conditions for each spot to be optimized independently. Overall, this 50-plex sandwich immunoassay represents the highest multiplexing level of a cross-reactivity-free antibody microarray achieved to-date. Furthermore, colocalized reagent delivery allows the assay to incorporate multiple antibody pairs that would otherwise cross-react when mixed together, overcoming a major limitation of other multiplexed assay formats29, and therefore the number of proteins to be measured could be further expanded.


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)

Fluorescent image of an assay slide and standard binding curves for 50 proteins simultaneously measured using the snap chip.(a) Fluorescent micrograph of a representative assay slide with 16 replicate arrays incubated in serial standard dilutions. (b) Close-up of the array within the dashed box in (a), with 153 spots and a 400 μm center-to-center spacing. Scale bar: 2 mm. (c) Standard curves for 50 proteins (for CA 15-3 the concentration is in unit/mL). Error bars represent standard deviations calculated from three independent experiments. Negative control fluorescence signals generated with 0 ng/mL protein concentrations are shown as separate data points.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Fluorescent image of an assay slide and standard binding curves for 50 proteins simultaneously measured using the snap chip.(a) Fluorescent micrograph of a representative assay slide with 16 replicate arrays incubated in serial standard dilutions. (b) Close-up of the array within the dashed box in (a), with 153 spots and a 400 μm center-to-center spacing. Scale bar: 2 mm. (c) Standard curves for 50 proteins (for CA 15-3 the concentration is in unit/mL). Error bars represent standard deviations calculated from three independent experiments. Negative control fluorescence signals generated with 0 ng/mL protein concentrations are shown as separate data points.
Mentions: To demonstrate the multiplexing capabilities of the snap chip using the double transfer method, an antibody colocalization microarray targeting breast cancer biomarkers252627, cancer-related proteins, and cytokines28 was performed according to a protocol outlined in Fig. 2. The immunoassay consisted of a total of 50 different antibodies and GFP calibrations spots, spotted in triplicate per pad, resulting in 2448 spots per slide. Aminosilane functionalized slides were used as transfer slides for the cAbs and dAbs. These slides act as good transfer surfaces due to their moderate contact angle, which helps to widen spots and therefore facilitate alignment, while on the other hand resulting in high droplets extending tens of micrometers above the surface, so as to ensure contact with the assay slide. The assay slides were coated with ~12 μm thick nitrocellulose pads, which provided a large binding capacity for cAbs and also facilitated the subsequent transfer of dAb droplets by partially absorbing the liquid. After spotting and transferring the cAb, the assay slide and the transfer slide with dAbs were stored in a −20 °C freezer. To run an assay, the slides were first rehydrated as described in detail in the methods section. Seven serial dilutions of each protein and a negative control (no protein) were incubated in the eight wells of each column of nitrocellulose pads on the assay slide, Fig. 5a. Variations in spot sizes between triplicate spots for antibody pairs, which match across all array pads, can be attributed to volumetric errors in the inkjet spotting, depending on the spotted antibody, Fig. 5b. The variations in spot size did not affect immunoassay data analysis because of the homogeneity across all the pads, and might be mitigated with larger volumes for each spot. The fluorescence intensity was quantified, and standard curves were generated, and are grouped based on the binding ranges of each antibody pair and the signal intensity, Fig. 5c. The LODs of 35 proteins were found to be in the pg/mL range, with the lowest being 3.3 pg/mL for CCL3, SI Table 2. The LOD might be improved by further optimizing the assay and storage conditions, since the current ACM format allows the conditions for each spot to be optimized independently. Overall, this 50-plex sandwich immunoassay represents the highest multiplexing level of a cross-reactivity-free antibody microarray achieved to-date. Furthermore, colocalized reagent delivery allows the assay to incorporate multiple antibody pairs that would otherwise cross-react when mixed together, overcoming a major limitation of other multiplexed assay formats29, and therefore the number of proteins to be measured could be further expanded.

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