General methods for quantitative interpretation of results of digital variable-volume assays

Abstract

In digital assays, devices typically require precisely controlled volumes since variation can cause biases in concentration estimates. Here, we develop methods to correct bias when compartment volumes are variable.

Wash-Free, Digital Immunoassay in Polydisperse Droplets

“…In the ideal scenario, the probability a certain droplet turns ON depends on analyte concentration (λ) and the volume distribution characterized by separately measured volumes ( v i ) (eq ). In such case, the analyte concentration can be readily calculated from experimental results without specific assumptions about the function that describes the droplet volume distribution . In other words, instead of having to assign a close-formed expression , to describe the volume distribution and finding the parameters from v i values ( i = 1,2, ..., m ), one can use v i values individually, and directly, without binning.…”

“…An advantage of digital assays over bulk assays is the ability to avoid calibration usually required to account for variation in the end signal, thanks to the ON/OFF nature of the signals of digital assays. In digital PCR assays, calibration is typically not necessary because the reactions happen in a close-to-ideal fashion, and the Poisson model can be used to calculate concentrations from the observed signals, even with large volume variations . However, in digital immunoassays implemented in the system described here and in others, the number of droplets with positive signals may not always be equal to the number of droplets containing one or more target entities.…”

“…In digital PCR assays, calibration is typically not necessary because the reactions happen in a close-to-ideal fashion, and the Poisson model can be used to calculate concentrations from the observed signals, even with large volume variations. 43 However, in digital immunoassays implemented in the system described here and in others, 48 the number of droplets with positive signals may not always be equal to the number of droplets containing one or more target entities. Therefore, it is important to perform the initial characterization of the parameters describing nonideal behaviors (ω, ϵ, and η) and premeasured volumes (v i ) (eq 2), but subsequent assay runs do not require their own standard curves.…”

“…In such case, the analyte concentration can be readily calculated from experimental results without specific assumptions about the function that describes the droplet volume distribution. 43 In other words, instead of having to assign a close-formed expression 44,45 to describe the volume distribution and finding the parameters from v i values (i = 1,2, ...,m), one can use v i values individually, and directly, without binning. These v i values are obtained in a manner that is separate from the assay and used for the calculation of the input concentration from each assay as constant.…”

A Portable Digital Loop-Mediated Isothermal Amplification Platform Based on Microgel Array and Hand-Held Reader

“…For a sample with a certain volume, larger number of partitions can produce more chambers for single DNA amplification, indicating that the digital NA amplification chip with abundant partitions can work in a wide range of DNA concentrations. Furthermore, if the chip has abundant partitions, its broader dynamic detection range can reduce the dilution ratio, thus eliminating the experimental error . According to these, we designed a chromium photomask for patterning microgel arrays with different microgel diameters of 100, 50, and 25 μm, respectively.…”