Scalable Signature-Based Molecular Diagnostics Through On-chip Biomarker Profiling Coupled with Machine Learning.

Molecular diagnostics have traditionally relied on discrete biological substances as diagnostic markers. In recent years however, advances in on-chip biomarker screening technologies and data analytics have enabled signature-based diagnostics. Such diagnostics aim to utilize unique combinations of multiple biomarkers or diagnostic 'fingerprints' rather than discrete analyte measurements. This approach has shown to improve both diagnostic accuracy and diagnostic specificity. In this review, signature-based diagnostics enabled by microfluidic and micro-/nano- technologies will be reviewed with a focus on device design and data analysis pipelines and methodologies. With increasing amounts of data available from microfluidic biomarker screening, isolation, and detection platforms, advanced data handling and analytics approaches can be employed. Thus, current data analysis approaches including machine learning and recent advances with image processing, along with potential future directions will be explored. Lastly, the needs and gaps in current literature will be elucidated to inform future efforts towards development of molecular diagnostics and biomarker screening technologies.

Scalable COVID-19 Detection Enabled by Lab-on-Chip Biosensors.

Introduction: The emergence of a novel coronavirus, SARS-CoV-2, has highlighted the need for rapid, accurate, and point-of-care diagnostic testing. As of now, there is not enough testing capacity in the world to meet the stated testing targets, which are expected to skyrocket globally for broader testing during reopening. Aim: This review focuses on the development of lab-on-chip biosensing platforms for diagnosis of COVID-19 infection. Results: We discuss advantages of utilizing lab-on-chip technologies in response to the current global pandemic, including their potential for low-cost, rapid sample-to-answer processing times, and ease of integration into a range of healthcare settings. We then highlight the development of magnetic, colorimetric, plasmonic, electrical, and lateral flow-based lab-on-chip technologies for the detection of SARS-CoV-2, in addition to other viruses. We focus on rapid, point-of-care technologies that can be deployed at scale, as such devices could be promising alternatives to the current gold standard of reverse transcription-polymerase chain reaction (RT-PCR) diagnostic testing. Conclusion: This review is intended to provide an overview of the current state-of-the-field and serve as a resource for innovative development of new lab-on-chip assays for COVID-19 detection.

Microfluidic enrichment of bacteria coupled to contact-free lysis on a magnetic polymer surface for downstream molecular detection.

We report on a microsystem that couples high-throughput bacterial immunomagnetic capture to contact-free cell lysis using an alternating current magnetic field (AMF) to enable downstream molecular characterization of bacterial nucleic acids. Traditional methods for cell lysis rely on either dilutive chemical methods, expensive biological reagents, or imprecise physical methods. We present a microchip with a magnetic polymer substrate (Mag-Polymer microchip), which enables highly controlled, on-chip heating of biological targets following exposure to an AMF. First, we present a theoretical framework for the quantitation of power generation for single-domain magnetic nanoparticles embedded in a polymer matrix. Next, we demonstrate successful bacterial DNA recovery by coupling (1) high-throughput, sensitive microfluidic immunomagnetic capture of bacteria to (2) on-chip, contact-free bacterial lysis using an AMF. The bacterial capture efficiency exceeded 76% at 50 ml/h at cell loads as low as ∼10 CFU/ml, and intact DNA was successfully recovered at starting bacterial concentrations as low as ∼1000 CFU/ml. Using the presented methodology, cell lysis becomes non-dilutive, temperature is precisely controlled, and potential contamination risks are eliminated. This workflow and substrate modification could be easily integrated in a range of micro-scale diagnostic systems for infectious disease.

Advances in diagnostic microfluidics.

Microfluidics is an emerging field in diagnostics that allows for extremely precise fluid control and manipulation, enabling rapid and high-throughput sample processing in integrated micro-scale medical systems. These platforms are well-suited for both standard clinical settings and point-of-care applications. The unique features of microfluidics-based platforms make them attractive for early disease diagnosis and real-time monitoring of the disease and therapeutic efficacy. In this chapter, we will first provide a background on microfluidic fundamentals, microfluidic fabrication technologies, microfluidic reactors, and microfluidic total-analysis-systems. Next, we will move into a discussion on the clinical applications of existing and emerging microfluidic platforms for blood analysis, and for diagnosis and monitoring of cancer and infectious disease. Together, this chapter should elucidate the potential that microfluidic systems have in the development of effective diagnostic technologies through a review of existing technologies and promising directions.

Microfluidics-Based Organism Isolation from Whole Blood: An Emerging Tool for Bloodstream Infection Diagnosis.

The diagnosis of bloodstream infections presents numerous challenges, in part, due to the low concentration of pathogens present in the peripheral bloodstream. As an alternative to existing time-consuming, culture-based diagnostic methods for organism identification, microfluidic devices have emerged as rapid, high-throughput and integrated platforms for bacterial and fungal enrichment, detection, and characterization. This focused review serves to highlight and compare the emerging microfluidic platforms designed for the isolation of sepsis-causing pathogens from blood and suggest important areas for future research.

Evaluation of different adsorbent materials for the untargeted and targeted bacterial VOC analysis using GC×GC-MS.

The analysis of bacterial volatile organic compounds has gained attraction as a non-invasive way to identify disease-causing organisms, given that bacteria have unique metabolisms and volatile metabolic byproducts. In the present research, different adsorbent materials (Carbopack Y, X, B, Carboxen 1000 and Tenax TA), packed singularly or in combination, were compared in terms of sampling performance (sensitivity, repeatability and selectivity) for the extraction of standards and bacterial volatile metabolites in vitro (from Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli). After extraction, bacterial volatile organic compounds were desorbed and analyzed in a comprehensive two-dimensional gas chromatography system coupled to a time-of-flight mass spectrometer (GC × GC-ToF MS). The results show that Tenax has the greater ability to extract the standard mix as well as volatile organic compounds with better repeatability (4-26 RSD%), higher sensitivity (on average ∼24 fold) compared to Carbopack Y, X and Carboxen 1000 tube, which followed in terms of performance. In addition, Tenax confirmed the best sensitivity and discriminatory power with no misclassification in the untargeted and unsupervised analysis for the differentiation of the bacterial species.

Printable QR code paper microfluidic colorimetric assay for screening volatile biomarkers.

We present a QR code paper microfluidic colorimetric assay that can exploit the hardware and software on mobile devices, and circumvent sample preparation by directly targeting volatile biomarkers. Our platform is a printable microarray of well-defined reaction regions, which outputs an instant diagnosis by directing the user to a URL containing their test result, while simultaneously storing epidemiological data for remote access and bioinformatics. To assist in the rapid identification of Escherichia coli in bloodstream infections, we employed an existing colorimetric reagent (p-dimethylaminocinnamaldehyde) and adapted its use to detect volatile indole, a biomarker produced by E. coli. Our assay was able to quantitatively detect indole in the headspace of E. coli culture after 12 h of growth (27.0 ±â€¯3.1 ppm), assisting in species-level identification hours earlier than existing methods. Results were confirmed with headspace solid-phase microextraction (HS-SPME) two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-ToFMS), which estimated indole concentration in E. coli culture to average 32.3 ±â€¯5.2 ppm after 12 h of growth. This QR paper microfluidic platform represents a novel development in both telemedicine and diagnostics using volatile biomarkers. We envision that our QR code platform can be extended to other colorimetric assays for real-time diagnostics in low-resource environments.

Combined immunomagnetic capture coupled with ultrasensitive plasmonic detection of circulating tumor cells in blood.

We demonstrate enhanced on-chip circulating tumor cell (CTC) detection through the incorporation of plasmonic-enhanced near-infrared (NIR) fluorescence screening. Specifically, the performance of plasmonic gold coated chips was evaluated on our previously reported immunomagnetic CTC capture system and compared to the performance of a regular chip. Three main performance metrics were evaluated: capture efficiency, capture reproducibility, and clinical efficacy. Use of the plasmonic chip to capture SK-BR-3 cells in PBS, resulted in a capture efficiency of 82%, compared to 76% with a regular chip. Both chips showed excellent capture reproducibility for all three cells lines evaluated (MCF-7, SK-BR-3, Colo 205) in both PBS and peripheral blood, with R2 values ranging from 0.983 to 0.996. Finally, performance of the plasmonic chip was evaluated on thirteen peripheral blood samples in patients with both breast and prostate cancer. The regular chip detected 2-8 cells per 5 mL of blood, while the plasmonic chip detected 8-85 cells per 5 mL of blood in parallel samples. In summary, we successfully demonstrate improved CTC capture and detection capabilities through use of plasmonic-enhanced near-infrared (NIR) fluorescence screening in both in vitro and ex vivo experiments. This work not only has the potential to improve clinical outcomes though improved CTC analysis, but also demonstrates successful interface design between plasmonic materials and cell capture for bioanalytical applications.

The volatile molecular profiles of seven Streptococcus pneumoniae serotypes.

In this study, the volatile molecule profile of Streptococcus pneumoniae serotypes was evaluated using solid phase microextraction (SPME) and two dimensional gas chromatography time-of-flight mass spectrometry (GC × GC-TOFMS). Here, seven serotypes (6B, 14, 15, 18C, 19F, 9V, and 23F) were analyzed in an isogenic background. We identified 13 core molecules associated with all seven serotypes, and seven molecules that were differentially produced between serotypes. Serotype 14 was found to have the most distinct volatile profile, and could be discriminated from the other six serotypes in aggregate with an area under the curve (AUC) of 89%. This study suggests that molecules from S. pneumoniae culture headspace show potential for rapid serotype identification.

Comprehensive volatile metabolic fingerprinting of bacterial and fungal pathogen groups.

The identification of pathogen-specific volatile metabolic 'fingerprints' could lead to the rapid identification of disease-causing organisms either directly from ex vivo patient bio-specimens or from in vitro cultures. In the present study, we have evaluated the volatile metabolites produced by 100 clinical isolates belonging to ten distinct pathogen groups that, in aggregate, account for 90% of bloodstream infections, 90% of urinary tract infections, and 80% of infections encountered in the intensive care unit setting. Headspace volatile metabolites produced in vitro were concentrated using headspace solid-phase microextraction and analyzed via two-dimensional gas chromatography time-of-flight mass spectrometry (HS-SPME-GC×GC-TOFMS). A total of 811 volatile metabolites were detected across all samples, of which 203 were: (1) detected in 9 or 10 (of 10) isolates belonging to one or more pathogen groups, and (2) significantly more abundant in cultures relative to sterile media. Network analysis revealed a distinct metabolic fingerprint associated with each pathogen group, and analysis via Random Forest using leave-one-out cross-validation resulted in a 95% accuracy for the differentiation between groups. The present findings support the results of prior studies that have reported on the differential production of volatile metabolites across pathogenic bacteria and fungi, and provide additional insight through the inclusion of pathogen groups that have seldom been studied previously, including Acinetobacter spp., coagulase-negative Staphylococcus, and Proteus mirabilis, as well as the utilization of HS-SPME-GC×GC-TOFMS for improved sensitivity and resolution relative to traditional gas chromatography-based techniques.

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice.

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15-1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.

An affordable open-source turbidimeter.

Turbidity is an internationally recognized criterion for assessing drinking water quality, because the colloidal particles in turbid water may harbor pathogens, chemically reduce oxidizing disinfectants, and hinder attempts to disinfect water with ultraviolet radiation. A turbidimeter is an electronic/optical instrument that assesses turbidity by measuring the scattering of light passing through a water sample containing such colloidal particles. Commercial turbidimeters cost hundreds or thousands of dollars, putting them beyond the reach of low-resource communities around the world. An affordable open-source turbidimeter based on a single light-to-frequency sensor was designed and constructed, and evaluated against a portable commercial turbidimeter. The final product, which builds on extensive published research, is intended to catalyze further developments in affordable water and sanitation monitoring.

Decontamination options for Bacillus anthracis-contaminated drinking water determined from spore surrogate studies.

Five parameters were evaluated with surrogates of Bacillus anthracis spores to determine effective decontamination alternatives for use in a contaminated drinking water supply. The parameters were as follows: (i) type of Bacillus spore surrogate (B. thuringiensis or B. atrophaeus), (ii) spore concentration in suspension (10(2) and 10(6) spores/ml), (iii) chemical characteristics of the decontaminant (sodium dichloro-S-triazinetrione dihydrate [Dichlor], hydrogen peroxide, potassium peroxymonosulfate [Oxone], sodium hypochlorite, and VirkonS), (iv) decontaminant concentration (0.01% to 5%), and (v) exposure time to decontaminant (10 min to 1 h). Results from 138 suspension tests with appropriate controls are reported. Hydrogen peroxide at a concentration of 5% and Dichlor or sodium hypochlorite at a concentration of 2% were highly effective at spore inactivation regardless of spore type tested, spore exposure time, or spore concentration evaluated. This is the first reported study of Dichlor as an effective decontaminant for B. anthracis spore surrogates. Dichlor's desirable characteristics of high oxidation potential, high level of free chlorine, and a more neutral pH than that of other oxidizers evaluated appear to make it an excellent alternative. All three oxidizers were effective against B. atrophaeus spores in meeting the EPA biocide standard of greater than a 6-log kill after a 10-min exposure time and at lower concentrations than typically reported for biocide use. Solutions of 5% VirkonS and Oxone were less effective as decontaminants than other options evaluated in this study and did not meet the EPA's efficacy standard for a biocide, although they were found to be as effective for concentrations of 10(2) spores/ml. Differences in methods and procedures reported by other investigators make quantitative comparisons among studies difficult.