Abstract

McCalla

Laboratory-based diagnostic technologies are advancing rapidly, which in turn improves patient care and health outcomes. These advances are largely concentrated in centralized laboratory facilities, however, and access to this new technology is limited in the developing world. New diagnostic technologies are often expensive, which also limits the use of these technologies based on healthcare costs. There is therefore a large gap between current technology and clinical need for simple, robust, and inexpensive assays. 

Many have attempted to close this gap with rapid, one-step molecular detection chemistries for biomarkers such as proteins, DNA, and RNA. While these technologies are promising, they sacrifice robustness and reproducibility for gains in speed, simplicity, and sensitivity.  Robustness and reproducibility are critical, particularly when assays are performed at the point of care in varying environmental conditions by individuals without laboratory training. Poor specificity could cause false positives, leading to expensive, unnecessary, and possibly harmful interventions. We propose a new molecular detection toolbox for disease diagnostics at the point of care based on a chemical amplification switch. Switch-like behavior is commonly observed in biological systems, and it is widely accepted that its function is to filter out non-specific signals. The proposed amplification switch will convert a specific target molecule into a high concentration DNA signal, producing an “on” state. Non-target molecules will produce a minimal DNA signal, and thus remain in an “off” state. Non-specific false positives can therefore be suppressed by maintaining the DNA signal in the “off” state. During the pilot stage of this project, we will develop chemistries to trigger switch turn-on using clinically relevant biomarkers such as RNA, DNA, and proteins. Ultimately, we aim to bring rapid and robust molecular diagnostics to all patients, regardless of location or economic status.

Significance

Biomarker detection is a vital step for treatment of a variety of maladies, from infectious disease to traumatic brain injury. A diagnostic test for malaria that requires no laboratory infrastructure would save ~2.2 million adjusted lives and prevent ~447 million unnecessary treatments per year. Rapid, on-site testing is unavailable for the vast majority of biomarkers in rural and limited resource settings2, creating a large gap between current technology and clinical needs. Sites that lack cold storage or shipping face additional barriers to accessible healthcare, as clinical samples may degrade in transit. Fixing these health disparities is a non-trivial challenge: the constraints on these tests are difficult to maintain outside of central laboratories. The technical requirements for running the assay must be simple, so that specialized training is not required to maintain testing capabilities. Additionally, they must be inexpensive, robust, and rapid (<1 hour).  Current assays to detect these molecules have a trade-off between complexity, speed, and robustness. If an assay to detect a dilute biomolecule is rapid or simple, it is not robust to environmental changes or operator error. Point-of-care diagnostics produce immediate results outside of advanced healthcare infrastructure and enables efficient healthcare delivery in rural areas. The long-term objective of the proposed work is a simple, rapid, robust assay to detect biomarkers outside of clinical laboratories at the point-of-care. 

We have engineered a biologically-inspired reaction to avoid false positives while maintaining speed and sensitivity. Switch-like behavior is commonly observed in biological systems. It is widely accepted that the function of these switches is filtering out noise. We propose to use a similar mechanism to turn off signal for low affinity, non-specific reactions. We have developed a tunable biochemical reaction with biphasic response to a reporter DNA molecule.  When the input DNA molecule is below a threshold concentration (phase I), the switch has a low output of reporter DNA. When the reporter DNA exceeds a threshold concentration (phase II), the reaction switches to a high reporter DNA output.  Therefore maintaining the reporter DNA in phase I will prevent a false positive signal. These reactions are rapid (<1 hour), one-step, and require no thermal cycling. 

The goal of the proposed work is to investigate the switch-based reaction for the detection of clinically relevant targets (Figure 1). We are currently detecting small DNA and RNA using this chemistry, and investigating methods to robustly maintain the reaction in phase I when a specific biomarker is not present. During the one year pilot program, we propose to expand the biochemistry shown above to detect genomic DNA, mRNA, and proteins. The impact of the switch-based assay will dramatically increase by expanding the pool of detectable diagnostic targets.  Detecting a wide variety of biomarkers will also advance the multiplexing capabilities of the assay. Detecting multiple diseases with similar symptoms, such as diarrheal diseases, is an important tool for clinicians. The proposed work is a key step in bringing life-saving diagnostic tests to underserved patient populations. The one-year pilot program will advance towards this goal by accomplishing the following aims.

Specific Aims

  1. Build a transduction chemistry for a variety of biomolecules. We will find optimal conditions and kinetics to transduce RNA, DNA, and proteins into short DNA triggers that can turn the chemical switch on. 
  2. Validate chemistry on clinically important biomarkers in complex samples. We will initiate switch turn-on with clinically significant biomolecules such as Pseudomonas aeruginosa surface antigens and genomic DNA in the presence of non-specific protein and DNA molecules.

Primary Contact

Stephanie McCalla stephanie.mccalla@montana.edu