Part 2: Where Are All the Multiplexed LFI’s?

By August 28, 2017Articles
Rapid diagnostic test, lateral flow assay, lateral flow test, point-of-care diagnostic

– Brendan O’Farrell, DCN Diagnostics

In part 1 of this article, I discussed some of the general factors impacting the development of multiplexed lateral flow assays and some of the approaches that are taken to design of these systems. One potential approach to multiplexing involves moving away from a standard line-based architecture and using an spot-array approach where the developed features are spots rather than lines. At DCN, we utilize a process called SymbolicsTM, which involves the pixilation of binding reagents using high precision dispensers to create arrays of spots in the lateral flow field. This approach brings potential in a number of ways:

1. Spot arrays can improve quantification and allow for dense multiplexing.

There is a growing requirement in point of need diagnostics for the generation of assays that can detect more than one analyte in a single device.  In a standard configuration, this means dispensing multiple lines perpendicular to the flow direction, separated by distances of 1 or more millimeters.  A typical issue seen in multiplexed assays of this nature is “line bleed.”  This condition occurs when the signal generated on one line merges with the next line resulting in the formation of background in the device, which lowers the sensitivity of the assay and can result in false positives.

Quantification in multiplexed assays is even more difficult due to the fact that the dynamics varies from assay to assay depending on their location on the strip.  Lateral flow assays are extremely time sensitive assays.  The reaction begins as soon as the sample and conjugate mix in the conjugate or sample pad.  The mixing continues during migration through the device to the test and control lines.  The reaction at the test line occurs quickly, typically in less than 30 seconds.  The flow rate of the reactants through the device can be extremely important to the performance of the assay.  Flow rate through an analytical membrane, typically nitrocellulose decreases in a non-linear fashion with distance from the origin.  As a result, the time taken for the reaction on first capture line versus the last capture line can be significantly different.  This has implications for the ability to generate quantitative assays in multiplexed formats. These issues can be overcome by using spot arrays in lateral flow fields rather than lines.

One of the most powerful potential applications of the SymbolicsTM lateral flow arraying process is the generation of multiplexed arrays for different analytes on a single strip with the added power of generating internal controls and replicates. This approach brings huge potential to increase the power of the lateral flow format.

2. Spot arrays allow us to generate alpha-numerical symbols instead of lines.

The formation of letters, symbols and lines in any orientation other than perpendicular to the direction of flow in a lateral flow assay is made difficult by the dynamics of flow and conjugate binding in the strip.  However, these difficulties can be demonstrably overcome by pixelating the dispensed reagents into individual small dots, spaced appropriately.

The patented SymbolicsTM technology that we use at DCN is based on the concept of the pixilation of reagents on the analytical membrane in a fashion that allows for the even development of each pixel. This appears, at first glance, to be a rather simple concept similar to dot matrix printing. The issue in lateral flow however, is that the formation of one feature in a flow path causes flow perturbations in the system that prevents the even formation of features behind it, or at least that is the general understanding. The key to the pixilation approach is in the controlled dispensing of the reagent spots for size and pitch (the center-to-center distance between spots in any axis).  These factors must be balanced for each reagent based on the binding characteristics of the reagents. When well optimized and controlled, it is possible to position individual spots of the correct size and pitch in the flow path of a lateral flow assay in such a way that each feature develops evenly and, most importantly, does not prevent the development of the other features surrounding it. With even development of individual features, it becomes possible to reproducibly build larger patterns in a lateral flow field.

SymbolicsTM, Practically Speaking

The shift to a spot array based process, which effectively represents a digital approach to feature creation in lateral flow fields, will represent a major shift in thinking for lateral flow developers and manufacturers.  In latter years, there has been a growing awareness of the capability of the lateral flow system in diverse markets, from commercial applications that have a requirement for simplicity in use and interpretation, to very highly specified quantitative and multiplexed biomarker panels. Although these applications require high performance from the assays, they are produced using relatively standard, although highly controlled processes. SymbolicsTM technology takes the requirement for process control to the next level, melding the lessons acquired in years of protein array development with the high speed, high throughput manufacturing approaches used in standard lateral flow production. Manufacturers will have some mental hurdles to cross in adopting this technology, as will assay developers. However, the potential application advantage that SymbolicsTM can bring is huge.

The first challenges will come in optimization. As mentioned previously, the key to even macro feature generation is the ability to develop micro features in the flow field that do not perturb the flow of liquid and conjugate and prevent even formation of the next feature in the pathway. The ability to do this depends on the size of the micro feature and the distance between the features, but also the binding affinity of the reagents. Each reagent therefore requires some optimization for feature size, reagent concentration and feature spacing. In most lateral flow applications, when screening for reagents, the focus tends to be on isolating antibodies with high on-rates (Kon). This remains true for SymbolicsTM. Features are small, so reagent on-rates must be high to ensure adequate sensitivity. Balancing that, the small size of the features means that it may be possible to reduce the amount of binding reagent significantly, resulting in savings in reagent costs. Materials must likewise be carefully specified and screened in order to best optimize performance in the SymbolicsTM format, however this is not significantly different to a standard lateral flow approach. Overall, the development approach is similar between the SymbolicsTM format and the standard lateral flow format. The major difference between the approach to optimization, development and manufacturing of the SymbolicsTM format is in the dispensing of capture reagents. Optimization and manufacturing requires high quality, precision dispensing systems, and levels of process control that are not typical in standard lateral flow applications.

 

Conclusion

Lateral flow is a continuously evolving format. Multiplexing is an obvious evolutionary step, allowing for a whole new swathe of potential applications. However, applications being considered should be carefully researched in terms of market need, regulatory complexity, reimbursement conditions as well as for the appropriate technical assay architecture and format to pursue.

As one potential multiplexing format, spot arraying facilitates another step in the evolution of the lateral flow system. It has the potential to enable a variety of applications, including:

  • More intuitive, easy to interpret consumer assays
  • Better controlled quantitative assays
  • Multiplexed assays in a variety of formats, including array, parallel multiplexing and “thermometer style” formats
  • More complex pattern recognition based detection methods for high density arraying or for quantification

As such, this approach provides a means to differentiate products, improve performance and to give end users an improved, intuitive and less error-prone experience.

More information on the SymbolicsTM approach, applications and intellectual property portfolio surrounding this pixilation approach to lateral flow multiplexing is available at www.symbolicsdx.com. Please also contact us at info@dcndx.com for more information on our capabilities in regard to multiplexed lateral flow assay development and production.

This topic will also be presented and discussed in detail at the upcoming Advanced Lateral Flow Course 2017 (www.dcnevents.com), so please join us in San Diego in October!