A culture-free biphasic approach for sensitive and rapid detection of pathogens in dried whole-blood matrix.

Blood stream infections (BSIs) cause high mortality, and their rapid detection remains a significant diagnostic challenge. Timely and informed administration of antibiotics can significantly improve patient outcomes. However, blood culture, which takes up to 5 d for a negative result, followed by PCR remains the gold standard in diagnosing BSI. Here, we introduce a new approach to blood-based diagnostics where large blood volumes can be rapidly dried, resulting in inactivation of the inhibitory components in blood. Further thermal treatments then generate a physical microscale and nanoscale fluidic network inside the dried matrix to allow access to target nucleic acid. The amplification enzymes and primers initiate the reaction within the dried blood matrix through these networks, precluding any need for conventional nucleic acid purification. High heme background is confined to the solid phase, while amplicons are enriched in the clear supernatant (liquid phase), giving fluorescence change comparable to purified DNA reactions. We demonstrate single-molecule sensitivity using a loop-mediated isothermal amplification reaction in our platform and detect a broad spectrum of pathogens, including gram-positive methicillin-resistant and methicillin-susceptible Staphylococcus aureus bacteria, gram-negative Escherichia coli bacteria, and Candida albicans (fungus) from whole blood with a limit of detection (LOD) of 1.2 colony-forming units (CFU)/mL from 0.8 to 1 mL of starting blood volume. We validated our assay using 63 clinical samples (100% sensitivity and specificity) and significantly reduced sample-to-result time from over 20 h to <2.5 h. The reduction in instrumentation complexity and costs compared to blood culture and alternate molecular diagnostic platforms can have broad applications in healthcare systems in developed world and resource-limited settings.

Spatial mapping of cancer tissues by OMICS technologies.

Spatial mapping of heterogeneity in gene expression in cancer tissues can improve our understanding of cancers and help in the rapid detection of cancers with high accuracy and reliability. Significant advancements have been made in recent years in OMICS technologies, which possess the strong potential to be applied in the spatial mapping of biopsy tissue samples and their molecular profiling to a single-cell level. The clinical application of OMICS technologies in spatial profiling of cancer tissues is also advancing. The current review presents recent advancements and prospects of applying OMICS technologies to the spatial mapping of various analytes in cancer tissues. We benchmark the current state of the art in the field to advance existing OMICS technologies for high throughput spatial profiling. The factors taken into consideration include spatial resolution, types of biomolecules, number of different biomolecules that can be detected from the same assay, labeled versus label-free approaches, and approximate time required for each assay. Further advancements are still needed for the widespread application of OMICs technologies in performing fast and high throughput spatial mapping of cancer tissues as well as their effective use in research and clinical applications.

Culture-free biphasic approach for sensitive detection of Escherichia coli O157:H7 from beef samples.

Foodborne illnesses are a major threat to public health also leading to significant mortality and financial and reputational damage to industry. It is very important to detect pathogen presence in food products early, rapidly, and accurately to avoid potential outbreaks and economic loss. However, "gold standard" culture methods, including enrichment of pathogens, can take up to several days. Moreover, the food matrix often interferes with nucleic acid amplification methods of detection, requiring DNA extraction from the sample for successful molecular detection of pathogens. Here, we introduce a "biphasic" amplification method that can achieve high sensitivity detection with background noise from ground beef food samples without culture or other extraction methods in 2.5 h. Homogenized ground beef is dried resulting in an increase in porosity of the dried food matrix to allowing amplification enzymes and primers to access the target DNA and initiate the reaction within the dried food matrix. Using Loop Mediated Isothermal Amplification, we demonstrate the detection of 1-3 cfu of Escherichia coli bacteria in 30 mg of dried food matrix. Our approach significantly lowers the time to result to less than a few hours and have a pronounced impact on reduction of instrumentation complexity and costs.

Portable Pathogen Diagnostics Using Microfluidic Cartridges Made from Continuous Liquid Interface Production Additive Manufacturing.

Biomedical diagnostics based on microfluidic devices have the potential to significantly benefit human health; however, the manufacturing of microfluidic devices is a key limitation to their widespread adoption. Outbreaks of infectious disease continue to demonstrate the need for simple, sensitive, and translatable tests for point-of-care use. Additive manufacturing (AM) is an attractive alternative to conventional approaches for microfluidic device manufacturing based on injection molding; however, there is a need for development and validation of new AM process capabilities and materials that are compatible with microfluidic diagnostics. In this paper, we demonstrate the development and characterization of AM cartridges using continuous liquid interface production (CLIP) and investigate process characteristics and capabilities of the AM microfluidic device manufacturing. We find that CLIP accurately produces microfluidic channels as small as 400 µm and that it is possible to routinely produce fluid channels as small as 100 µm with high repeatability. We also developed a loop-mediated isothermal amplification (LAMP) assay for detection of E. coli from whole blood directly on the CLIP-based AM microfluidic cartridges, with a 50 cfu/µL limit of detection, validating the use of CLIP processes and materials for pathogen detection. The portable diagnostic platform presented in this paper could be used to investigate and validate other AM processes for microfluidic diagnostics and could be an important component of scaling up the diagnostics for current and future infectious diseases and pandemics.

Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Ultrasensitive Detection of SARS-CoV-2 in Saliva and Viral Transport Medium Clinical Samples.

The COVID-19 pandemic has underscored the shortcomings in the deployment of state-of-the-art diagnostics platforms. Although several polymerase chain reaction (PCR)-based techniques have been rapidly developed to meet the growing testing needs, such techniques often need samples collected through a swab, the use of RNA extraction kits, and expensive thermocyclers in order to successfully perform the test. Isothermal amplification-based approaches have also been recently demonstrated for rapid severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection by minimizing sample preparation while also reducing the instrumentation and reaction complexity. In addition, there are limited reports of saliva as the sample source, and some of these indicate inferior sensitivity when comparing reverse transcription loop-mediated isothermal amplification (RT-LAMP) with PCR-based techniques. In this paper, we demonstrate an improved sensitivity assay from saliva using a two-step RT-LAMP assay, where a short 10 min RT step is performed with only B3 and backward inner primers before the final reaction. We show that while the one-step RT-LAMP demonstrates satisfactory results, the optimized two-step approach allows detection of only few molecules per reaction and performs significantly better than the one-step RT-LAMP and conventional two-step RT-LAMP approaches with all primers included in the RT step. We show control measurements with RT-PCR, and importantly, we demonstrate RNA extraction-free RT-LAMP-based assays for detection of SARS-CoV-2 from viral transport media and saliva clinical samples.

COVID-19 Point-of-Care Diagnostics: Present and Future.

Point-of-care (POC) detection technologies that enable decentralized, rapid, sensitive, low-cost diagnostics of COVID-19 infection are urgently needed around the world. With many technologies approved for commercialization in the past 10 months, the field of COVID-19 POC diagnostics is rapidly evolving. In this Perspective, we analyze the current state of POC technologies for the diagnosis and monitoring of COVID-19 infection and discuss future challenges in COVID-19 diagnostics. As the COVID-19 pandemic becomes endemic, the advances gained during this past year will likely also be utilized for future prediction of emerging outbreaks and pandemics.

Rapid isothermal amplification and portable detection system for SARS-CoV-2.

The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay protocols and primer sequences become widely known, many laboratories perform diagnostic tests using methods such as RT-PCR or reverse transcription loop mediated isothermal amplification (RT-LAMP). Here, we report an RT-LAMP isothermal assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and demonstrate the assay on clinical samples using a simple and accessible point-of-care (POC) instrument. We characterized the assay by dipping swabs into synthetic nasal fluid spiked with the virus, moving the swab to viral transport medium (VTM), and sampling a volume of the VTM to perform the RT-LAMP assay without an RNA extraction kit. The assay has a limit of detection (LOD) of 50 RNA copies per µL in the VTM solution within 30 min. We further demonstrate our assay by detecting SARS-CoV-2 viruses from 20 clinical samples. Finally, we demonstrate a portable and real-time POC device to detect SARS-CoV-2 from VTM samples using an additively manufactured three-dimensional cartridge and a smartphone-based reader. The POC system was tested using 10 clinical samples, and was able to detect SARS-CoV-2 from these clinical samples by distinguishing positive samples from negative samples after 30 min. The POC tests are in complete agreement with RT-PCR controls. This work demonstrates an alternative pathway for SARS-CoV-2 diagnostics that does not require conventional laboratory infrastructure, in settings where diagnosis is required at the point of sample collection.

Simultaneous time-varying viscosity, elasticity, and mass measurements of single adherent cancer cells across cell cycle.

Biophysical studies on single cells have linked cell mechanics to physiology, functionality and disease. Evaluation of mass and viscoelasticity versus cell cycle can provide further insights into cell cycle progression and the uncontrolled proliferation of cancer. Using our pedestal microelectromechanical systems resonant sensors, we have developed a non-contact interferometric measurement technique that simultaneously tracks the dynamic changes in the viscoelastic moduli and mass of adherent colon (HT-29) and breast cancer (MCF-7) cells from the interphase through mitosis and then to the cytokinesis stages of their growth cycle. We show that by combining three optomechanical parameters in an optical path length equation and a two-degree-of-freedom model, we can simultaneously extract the viscoelasticity and mass as a function of the nano-scaled membrane fluctuation of each adherent cell. Our measurements are able to discern between soft and stiff cells across the cell cycle and demonstrated sharp viscoelastic changes due to cortical stiffening around mitosis. Cell rounding before division can be detected by measurement of mechanical coupling between the cells and the sensors. Our measurement device and method can provide for new insights into the mechanics of single adherent cells versus time.

Smartphone-based multiplex 30-minute nucleic acid test of live virus from nasal swab extract.

Rapid, sensitive and specific detection and reporting of infectious pathogens is important for patient management and epidemic surveillance. We demonstrated a point-of-care system integrated with a smartphone for detecting live virus from nasal swab media, using a panel of equine respiratory infectious diseases as a model system for corresponding human diseases such as COVID-19. Specific nucleic acid sequences of five pathogens were amplified by loop-mediated isothermal amplification on a microfluidic chip and detected at the end of reactions by the smartphone. Pathogen-spiked horse nasal swab samples were correctly diagnosed using our system, with a limit of detection comparable to that of the traditional lab-based test, polymerase chain reaction, with results achieved in ∼30 minutes.

Synchronized Electromechanical Shock Wave-Induced Bacterial Transformation.

We report a simple device that generates synchronized mechanical and electrical pressure waves for carrying out bacterial transformation. The mechanical pressure waves are produced by igniting a confined nanoenergetic composite material that provides ultrahigh pressure. Further, this device has an arrangement through which a synchronized electric field (of a time-varying nature) is initiated at a delay of ≈85 µs at the full width half-maxima point of the pressure pulse. The pressure waves so generated are incident to a thin aluminum-polydimethylsiloxane membrane that partitions the ignition chamber from the column of the mixture containing bacterial cells (Escherichia coli BL21) and 4 kb transforming DNA. A combination of mechanical and electrical pressure pulse created through the above arrangement ensures that the transforming DNA transports across the cell membrane into the cell, leading to a transformation event. This unique device has been successfully operated for efficient gene (∼4 kb) transfer into cells. The transformation efficacy of this device is found comparable to the other standard methods and protocols for carrying out the transformation.

Smartphone-imaged microfluidic biochip for measuring CD64 expression from whole blood.

Sepsis, a life-threatening syndrome that contributes to millions of deaths annually worldwide, represents a moral and economic burden to the healthcare system. Although no single, or even a combination of biomarkers has been validated for the diagnosis of sepsis, multiple studies have shown the high specificity of CD64 expression on neutrophils (nCD64) to sepsis. The analysis of elevated nCD64 in the first 2-6 hours after infection during the pro-inflammatory stage could significantly contribute to early sepsis diagnosis. Therefore, a rapid and automated device to periodically measure nCD64 expression at the point-of-care (POC) could lead to timely medical intervention and reduced mortality rates. Current accepted technologies for measuring nCD64 expression, such as flow cytometry, require manual sample preparation and long incubation times. For POC applications, however, the technology should be able to measure nCD64 expression with little to no sample preparation. In this paper, we demonstrate a smartphone-imaged microfluidic biochip for detecting nCD64 expression in under 50 min. In our assay, first unprocessed whole blood is injected into a capture chamber to immunologically capture nCD64 along a staggered array of pillars, which were previously functionalized with an antibody against CD64. Then, an image of the capture channel is taken using a smartphone-based microscope. This image is used to measure the cumulative fraction of captured cells (γ) as a function of length in the channel. During the image analysis, a statistical model is fitted to γ in order to extract the probability of capture of neutrophils per collision with a pillar (ε). The fitting shows a strong correlation with nCD64 expression measured using flow cytometry (R2 = 0.82). Finally, the applicability of the device to sepsis was demonstrated by analyzing nCD64 from 8 patients (37 blood samples analyzed) along the time they were admitted to the hospital. Results from this analysis, obtained using the smartphone-imaged microfluidic biochip were compared with flow cytometry. Again, a correlation coefficient R2 = 0.82 (slope = 0.99) was obtained demonstrating a good linear correlation between the two techniques. Deployment of this technology in ICU could significantly enhance patient care worldwide.

3D printing for preoperative planning and surgical training: a review.

Surgeons typically rely on their past training and experiences as well as visual aids from medical imaging techniques such as magnetic resonance imaging (MRI) or computed tomography (CT) for the planning of surgical processes. Often, due to the anatomical complexity of the surgery site, two dimensional or virtual images are not sufficient to successfully convey the structural details. For such scenarios, a 3D printed model of the patient's anatomy enables personalized preoperative planning. This paper reviews critical aspects of 3D printing for preoperative planning and surgical training, starting with an overview of the process-flow and 3D printing techniques, followed by their applications spanning across multiple organ systems in the human body. State of the art in these technologies are described along with a discussion of current limitations and future opportunities.

Robust label-free microRNA detection using one million ISFET array.

Detection of nucleic acid molecules is one of the most pervasive assays in biology, medicine, and agriculture applications. Currently, most comely used DNA/RNA detection platforms use fluorescence labeling and require lab-scale setting for performing the assay. There is a need for developing less expensive, label-free, and rapid detection of biomolecules with minimal utilization of resources. Use of electrical approaches for detection of biomolecules by utilizing their inherent charge is a promising direction for biosensing assays. Here, we report a 1024 × 1024 array of Ion Sensitive Field Effect Transistors (ISFET) as label free sensors for detection of nucleic acid molecules. Using PNA probe functionalized on these ISFET array, we robustly detected miRNA Let-7b by measuring changes in drain current after hybridization of target molecules with concentration as low as 1 nM. We demonstrate that mismatched or non-complementary target molecules resulted in statistically smaller changes. Most importantly, the high-density sensor array shows unprecedented reliability and robustness with P values <0.0001 for all experiments. Practical implementation of this platform could have a wide range of applications in high-throughput nucleic acid genotyping, detection of amplified pathogenic nucleic acid, detection of cell-free DNA, and electrical readouts for current hybridization-based DNA biomolecular assays.

Mobile Platform for Multiplexed Detection and Differentiation of Disease-Specific Nucleic Acid Sequences, Using Microfluidic Loop-Mediated Isothermal Amplification and Smartphone Detection.

New tools are needed to enable rapid detection, identification, and reporting of infectious viral and microbial pathogens in a wide variety of point-of-care applications that impact human and animal health. We report the design, construction, and characterization of a platform for multiplexed analysis of disease-specific DNA sequences that utilizes a smartphone camera as the sensor in conjunction with a hand-held "cradle" that interfaces the phone with a silicon-based microfluidic chip embedded within a credit-card-sized cartridge. Utilizing specific nucleic acid sequences for four equine respiratory pathogens as representative examples, we demonstrated the ability of the system to utilize a single 15 µL droplet of test sample to perform selective positive/negative determination of target sequences, including integrated experimental controls, in approximately 30 min. Our approach utilizes loop-mediated isothermal amplification (LAMP) reagents predeposited into distinct lanes of the microfluidic chip, which when exposed to target nucleic acid sequences from the test sample, generates fluorescent products that when excited by appropriately selected light emitting diodes (LEDs), are visualized and automatically analyzed by a software application running on the smartphone microprocessor. The system achieves detection limits comparable to those obtained by laboratory-based methods and instruments. Assay information is combined with the information from the cartridge and the patient to populate a cloud-based database for epidemiological reporting of test results.

A microfluidic technique to estimate antigen expression on particles.

Antigen expression is an important biomarker for cell analysis and disease diagnosis. Traditionally, antigen expression is measured using a flow cytometer which, due to its cost and labor intensive sample preparation, is unsuitable to be used at the point-of-care. Therefore, an automatic, miniaturized assay which can measure antigen expression in the patient could aid in making crucial clinical decisions rapidly. Such a device would also expand the use of such an assay in basic research in biology. In this paper, we present a microfluidic device that can be used to measure antigen expression on cells. We demonstrate our approach using biotin-neutravidin as the binding pair using experimental and computational approaches. We flow beads with varying biotin surface densities (mr ) through a polydimethylsiloxane channel with cylindrical pillars functionalized with neutravidin. We analyze how shear stress and collision angle, the angle at which the beads collide with the pillars, affect the angular location of beads captured on the pillars. We also find that the fraction of captured beads as a function of distance (γ) in the channel is affected by mr . Using γ, we derive the probability of capture per collision with the pillar (ε). We show that ε is linearly related to mr , which is analogous to the expression level of proteins on cell surfaces. Although demonstrated with beads, this assay can next be expanded with cells, thus paving the way for a rapid antigen expression test.