5 Key Challenges of Molecular Assay Development & How to Overcome Them

The need for high-throughput, accurate molecular assay development is ever-increasing in all phases of drug development. With the higher demand for detecting infectious diseases, cancer mutations, and genetic conditions, in addition to finding the next blockbuster drug, researchers often face growing pressures to optimize molecular assay performance, cost, and regulatory compliance. This blog outlines five key challenges with molecular assay development, including sensitivity, specificity, scalability, reproducibility, and resource constraints, and highlight how precise automated liquid handling systems can be key to optimizing molecular assays and solving these technical challenges. 

1. Achieving High Sensitivity: Detecting Low-Abundance Targets

The Challenge

High sensitivity is crucial for detecting the lowest possible concentration of an analyte. This is vital during clinical diagnostics when detecting low-abundance targets like ctDNA or viral RNA^1,2. Accurate results are required to prevent false negatives, which can directly impact patient outcomes. Compound screening equally requires high sensitivity to ensure potential hits are not missed during early drug development stages³. 

The Solution

Precise handling and accurate dispensing of tiny volumes are essential for high sensitivity in molecular assay development. The I.DOT liquid handler and G.PURE NGS Clean-Up Device from DISPENDIX can aid in increasing sensitivity in multiple molecular assays, including polymerase chain reaction (PCR) and next-generation sequencing (NGS) workflows, with dispensing capabilities on a nanoliter scale, enabling dispensing with precision and accuracy.

2. Ensuring High Specificity: Reducing Background Noise

The Challenge

Along with the issue of false negatives, there is also the problem of cross-reactivity and false positives⁴. Assay specificity is the ability of an assay to correctly identify the target analytes without reacting to non-target molecules. Inaccurate dispensing introduces contaminants or causes cross-contamination of reagents during assay development, which can harm specificity. Specificity is essential during clinical diagnostics to ensure patients are correctly diagnosed and receive the proper treatment plans. Low specificity in R&D can lead to high background noise, which reduces the confidence in drug candidates and could lead to identifying candidates that may fail further down the drug development pipeline³.

The Solution

Non-contact dispensing methods, like those used by the I.DOT non-contact liquid dispenser can help eliminate the risks of false positives during molecular assay development with precise and accurate dispensing, ensuring confidence and reliability in your results (Fig. 1). 

Read our  full-length article to learn more about how high sensitivity and specificity are essential in diagnostic assay development, and how automated liquid handling systems can provide accurate and precise dispensing.

Figure 1. The I.DOT liquid handler from DISPENDIX offers automated, non-contact, precision dispensing, enabling sensitivity and specificity during molecular assay development. 

3. Optimizing Limited Reagents and Samples: Miniaturization

The Challenge

Researchers often work with expensive reagents, including high-quality monoclonal primary antibodies in ELISA workflows⁵. Ensuring workflows preserve reagents and are cost-effective can be incredibly difficult. To add to the pressures of research in a clinical setting, workflows often involve using limited or precious patient samples, therefore, ensuring enough material is available for future or repeated analyses can be challenging.

The Solution

The best way to combat issues involving preservation of reagents and clinical samples is by assay miniaturization, which significantly reduces volumes to conserve valuable reagents and samples⁶. The I.DOT liquid handler from DISPENDIX provides precision dispensing, with a dead volume of 1 microliter, the I.DOT conserves reagents by up to 50%, resulting in significantly reduced volumes of reagents and samples needed. Utilizing an automated liquid handling system like the I.DOT during molecular assay development can lead to considerable cost savings through scaling down, without compromising performance and high-quality results. 

4. Achieving Scalability with Speed and Precision

The Challenge

One of the most challenging aspects of molecular assay development is screening for numerous potential hits across various conditions while avoiding the burden of time-consuming manual pipetting. With manual workflows, researchers struggle with lengthy assay times and increased human errors^7,8. Often, researchers are left to decide between throughput and compromising precision and accuracy.

The Solution

With advanced dispensing systems like the I.DOT Liquid Handler and G.PURE NGS Clean-Up Device from DISPENDIX, researchers don’t have to choose between high-throughput and reliable results. These instruments offer automated, precise liquid handling, which can accelerate research without compromising precision. The G.PREP NGS Bundle enables researchers to run thousands of samples daily at speed, ensuring high-throughput yet accurate results (Fig.2). 

Read our full-length article outlining how assay miniaturization and scaling up can accelerate drug discovery and how automated liquid handling systems can provide high-throughput yet precise and reliable results. 

5. Ensuring Reproducibility and High Quality Data

The Challenge

In R&D and clinical settings, consistent results are essential in molecular assay development to ensure reproducibility in peer-reviewed research and adherence to good manufacturing practice (GMP). However, manual workflows often introduce the risk of human error and inconsistencies between batches, particularly when handling low pipetting volumes^7,8. 

The Solution

The I.DOT liquid handler from DISPENDIX offers automated non-contact dispensing, ensuring consistent droplet sizes and placements, eliminating the chance for human errors. DISPENDIX platforms also support traceability, audit logging, and sample tracking which align with regulatory requirements.

Read our full-length article to explore how automated liquid handling systems can aid in assay optimization, ensuring results are reproducible, consistent, and abide by regulatory standards. 

Figure 2. The G.PREP bundle from DISPENDIX includes the I.DOT and G.PURE instruments, offering robust solutions for automating NGS workflows. This enables assay miniaturization down to 1/10th reaction volume and scalability, enabling high-throughput, accurate, and reproducible results. 

Conclusion: Build Stronger Assays by Addressing Core Development Challenges

Molecular assay development is complex and comes with its challenges, especially those related to assay sensitivity, specificity, scalability, reproducibility, and resource constraints. Automated liquid handling systems like the I.DOT and G.PURE from DISPENDIX can help overcome these challenges and optimize molecular assays through precision, high-throughput, and accurate dispensing. 

Download the I.DOT brochure or the G.PREP brochure today, and discover how automated liquid handling systems can help you overcome challenges with molecular assay development, giving you confidence in your assay workflows!

References

1. Chen S, Huang V, Xu X, et al. Widespread and Functional RNA Circularization in Localized Prostate Cancer. Cell. 2019;176(4):831-843.e22. doi:10.1016/j.cell.2019.01.025
2. Reville PK, Wang B, Marvin-Peek J, et al. Blood-Based Proteomic Profiling Identifies OSMR as a Novel Biomarker of AML Outcomes. Blood J. Published online April 3, 2025:blood.2024027244. doi:10.1182/blood.2024027244
3. Hughes J, Rees S, Kalindjian S, Philpott K. Principles of early drug discovery. Br J Pharmacol. 2011;162(6):1239-1249. doi:10.1111/j.1476-5381.2010.01127.x
4. Arevalo-Rodriguez I, Buitrago-Garcia D, Simancas-Racines D, et al. False-negative results of initial RT-PCR assays for COVID-19: A systematic review. Hozbor DF, ed. PLOS ONE. 2020;15(12):e0242958. doi:10.1371/journal.pone.0242958
5. Weller MG. Quality Issues of Research Antibodies. Anal Chem Insights. 2016;11:ACI.S31614. doi:10.4137/ACI.S31614
6. Carstens C, Elbracht R, Gärtner C, Becker H. Opportunities and limits of cell-based assay miniaturization in drug discovery. Expert Opin Drug Discov. 2010;5(7):673-679. doi:10.1517/17460441.2010.488264
7. Guan XL, Chang DPS, Mok ZX, Lee B. Assessing variations in manual pipetting: An under-investigated requirement of good laboratory practice. J Mass Spectrom Adv Clin Lab. 2023;30:25-29. doi:10.1016/j.jmsacl.2023.09.001
8. Lippi G, Lima-Oliveira G, Brocco G, Bassi A, Salvagno GL. Estimating the intra- and inter-individual imprecision of manual pipetting. Clin Chem Lab Med CCLM. 2017;55(7). doi:10.1515/cclm-2016-0810