High Sensitivity and High Throughput Magnetic Flow CMOS Cytometers with 2D Oscillator Array and Inter-Sensor Spectrogram Cross-correlation.

In the paper, we present an integrated flow cytometer with a 2D array of magnetic sensors based on dual-frequency oscillators in a 65-nm CMOS process, with the chip packaged with microfluidic controls. The sensor architecture and the presented array signal processing allows uninhibited flow of the sample for high throughput without the need for hydrodynamic focusing to a single sensor. To overcome the challenge of sensitivity and specificity that comes as a trade off with high throughout, we perform two levels of signal processing. First, utilizing the fact that a magnetically tagged cell is expected to excite sequentially an array of sensors in a time-delayed fashion, we perform inter-site cross-correlation of the sensor spectrograms that allows us to suppress the probability of false detection drastically, allowing theoretical sensitivity reaching towards sub-ppM levels that is needed for rare cell or circulating tumor cell detection. In addition, we implement two distinct methods to suppress correlated low frequency drifts of singular sensors-one with an on-chip sensor reference and one that utilizes the frequency dependence of the susceptibility of super-paramagnetic magnetic beads that we deploy as tags. We demonstrate these techniques on a 7×7 sensor array in 65 nm CMOS technology packaged with microfluidics with magnetically tagged dielectric particles and cultu lymphoma cancer cells.

A computer vision enhanced smart phone platform for microfluidic urine glucometry.

Glucose is an important biomarker for diagnosing and prognosing various diseases, including diabetes and hypoglycemia, which can have severe side effects, symptoms, and even lead to death in patients. As a result, there is a need for quick and economical glucose level measurements to help identify those at potential risk. With the increase in smartphone users, portable smartphone glucose sensors are becoming popular. In this paper, we present a disposable microfluidic glucose sensor that accurately and rapidly quantifies glucose levels in human urine using a combination of colorimetric analysis and computer vision. This glucose sensor implements a disposable microfluidic device based on medical-grade tapes and glucose analysis strips on a glass slide integrated with a custom-made polydimethylsiloxane (PDMS) micropump that accelerates capillary flow, making it economical, convenient, rapid, and equipment-free. After absorbing the target solution, the disposable device is slid into the 3D-printed main chassis and illuminated exclusively with Light Emitting Diode (LED) illumination, which is pivotal to color-sensitive experiments. After collecting images, the images are imported into the algorithm to measure the glucose levels using computer vision and average RGB values measurements. This article illustrates the impressive accuracy and consistency of the glucose sensor in quantifying glucose in sucrose water. This is evidenced by the close agreement between the computer vision method used by the sensor and the traditional method of measuring in the biology field, as well as the small variation observed between different sensor performances. The exponential regression curve used in the study further confirms the strong relationship between glucose concentrations and average RGB values, with an R-square value of 0.997 indicating a high degree of correlation between these variables. The article also emphasizes the potential transferability of the solution described to other types of assays and smartphone-based sensors.

Clinical evaluation of a fully electronic microfluidic white blood cell analyzer.

The White Blood Cell (WBC) count is one of the key parameters signaling the health of the immune system. Abnormal WBC counts often signal a systemic insult to the body such as an underlying infection or an adverse side effect to medication. Typically, the blood collected is sent to a central lab for testing, and results come back within hours, which is often inconvenient and may delay time-sensitive diagnosis or treatment. Here, we present the CytoTracker, a fully electronic, microfluidic based instant WBC analyzer with the potential to be used at point-of-care. The CytoTracker is a lightweight, portable, affordable platform capable of quantifying WBCs within minutes using only 50 µl of blood (approximately one drop of blood). In this study, we clinically evaluated the accuracy and performance of CytoTracker in measuring WBC and granulocyte counts. A total of 210 adult patients were recruited in the study. We validated the CytoTracker against a standard benchtop analyzer (Horiba Point of Care Hematology Analyzer, ABX Micros 60). Linear dynamic ranges of 2.5 k/µl- 35 k/µl and 0.6 k/µl- 26 k/µl were achieved for total WBC count and granulocyte count with correlation coefficients of 0.97 and 0.98. In addition, we verified CytoTracker's capability of identifying abnormal blood counts with above 90% sensitivity and specificity. The promising results of this clinical validation study demonstrate the potential for the use of the CytoTracker as a reliable and accurate point-of-care WBC analyzer.

Cell phone microscopy enabled low-cost manufacturable colorimetric urine glucose test.

Glucose serves as a pivotal biomarker crucial for the monitoring and diagnosis of a spectrum of medical conditions, encompassing hypoglycemia, hyperglycemia, and diabetes, all of which may precipitate severe clinical manifestations in individuals. As a result, there is a growing demand within the medical domain for the development of rapid, cost-effective, and user-friendly diagnostic tools. In this research article, we introduce an innovative glucose sensor that relies on microfluidic devices meticulously crafted from disposable, medical-grade tapes. These devices incorporate glucose urine analysis strips securely affixed to microscope glass slides. The microfluidic channels are intricately created through laser cutting, representing a departure from traditional cleanroom techniques. This approach streamlines production processes, enhances cost-efficiency, and obviates the need for specialized equipment. Subsequent to the absorption of the target solution, the disposable device is enclosed within a 3D-printed housing. Image capture is seamlessly facilitated through the use of a smartphone camera for subsequent colorimetric analysis. Our study adeptly demonstrates the glucose sensor's capability to accurately quantify glucose concentrations within sucrose solutions. This is achieved by employing an exponential regression model, elucidating the intricate relationship between glucose concentrations and average RGB (Red-Green-Blue) values. Furthermore, our comprehensive analysis reveals minimal variation in sensor performance across different instances. Significantly, this study underscores the potential adaptability and versatility of our solution for a wide array of assay types and smartphone-based sensor systems, making it particularly promising for deployment in resource-constrained settings and undeveloped countries. The robust correlation established between glucose concentrations and average RGB values, substantiated by an impressive R-square value of 0.98709, underscores the effectiveness and reliability of our pioneering approach within the medical field.

Advancing Healthcare: Synergizing Biosensors and Machine Learning for Early Cancer Diagnosis.

Cancer is a fatal disease and a significant cause of millions of deaths. Traditional methods for cancer detection often have limitations in identifying the disease in its early stages, and they can be expensive and time-consuming. Since cancer typically lacks symptoms and is often only detected at advanced stages, it is crucial to use affordable technologies that can provide quick results at the point of care for early diagnosis. Biosensors that target specific biomarkers associated with different types of cancer offer an alternative diagnostic approach at the point of care. Recent advancements in manufacturing and design technologies have enabled the miniaturization and cost reduction of point-of-care devices, making them practical for diagnosing various cancer diseases. Furthermore, machine learning (ML) algorithms have been employed to analyze sensor data and extract valuable information through the use of statistical techniques. In this review paper, we provide details on how various machine learning algorithms contribute to the ongoing development of advanced data processing techniques for biosensors, which are continually emerging. We also provide information on the various technologies used in point-of-care cancer diagnostic biosensors, along with a comparison of the performance of different ML algorithms and sensing modalities in terms of classification accuracy.

Multi-modal sensing with integrated machine learning to differentiate specific leukocytes targeted by electrically sensitive hybrid particles.

The growing need for personalized, accurate, and non-invasive diagnostic technology has resulted in significant advancements, from pushing current mechanistic limitations to innovative modality developments across various disease-related biomarkers. However, there still lacks clinical solutions for analyzing multiple biomarkers simultaneously, limiting prognosis for patients suffering with complicated diseases or comorbidities. Here, we conceived, fabricated, and validated a multifrequency impedance cytometry apparatus with novel frequency-sensitive barcoded metal oxide Janus particles (MOJPs) as cell-receptor targeting agents. These microparticles are modulated by a metal oxide semi-coating which exhibit electrical property changes in a multifrequency electric field and are functionalized to target CD11b and CD66b membrane proteins on neutrophils. A multi-modal system utilizing supervised machine learning and simultaneous high-speed video microscopy classifies immune-specific surface receptors targeted by MOJPs as they form neutrophil-MOJP conjugates, based on multivariate multifrequency electrical recordings. High precision and sensitivity were determined based on the type of MOJPs conjugated with cells (>90% accuracy between neutrophil-MOJP conjugates versus cells alone). Remarkably, the design could differentiate the number of MOJPs conjugated per cell within the same MOJP class (>80% accuracy); which also improved comparing electrical responses across different MOJP types (>75% accuracy) as well. Such trends were consistent in individual blood samples and comparing consolidated data across multiple samples, demonstrating design robustness. The configuration may further expand to include more MOJP types targeting critical biomarker receptors in one sample and increase the modality's multiplexing potential.

Digital assay for rapid electronic quantification of clinical pathogens using DNA nanoballs.

Fast and accurate detection of nucleic acids is key for pathogen identification. Methods for DNA detection generally rely on fluorescent or colorimetric readout. The development of label-free assays decreases costs and test complexity. We present a novel method combining a one-pot isothermal generation of DNA nanoballs with their detection by electrical impedance. We modified loop-mediated isothermal amplification by using compaction oligonucleotides that self-assemble the amplified target into nanoballs. Next, we use capillary-driven flow to passively pass these nanoballs through a microfluidic impedance cytometer, thus enabling a fully compact system with no moving parts. The movement of individual nanoballs is detected by a change in impedance providing a quantized readout. This approach is flexible for the detection of DNA/RNA of numerous targets (severe acute respiratory syndrome coronavirus 2, HIV, ß-lactamase gene, etc.), and we anticipate that its integration into a standalone device would provide an inexpensive (<$5), sensitive (10 target copies), and rapid test (<1 hour).

A two-minute assay for electronic quantification of antibodies in saliva enabled through a reusable microfluidic multi-frequency impedance cytometer and machine learning analysis.

The use of saliva as a diagnostic fluid has always been appealing due to the ability for rapid and non-invasive sampling for monitoring health status and the onset and progression of disease and treatment progress. Saliva is rich in protein biomarkers and provides a wealth of information for diagnosis and prognosis of various disease conditions. Portable electronic tools which rapidly monitor protein biomarkers would facilitate point-of-care diagnosis and monitoring of various health conditions. For example, the detection of antibodies in saliva can enable rapid diagnosis and tracking disease pathogenesis of various auto-immune diseases like sepsis. Here, we present a novel method involving immuno-capture of proteins on antibody coated beads and electrical detection of dielectric properties of the beads. The changes in electrical properties of a bead when capturing proteins are extremely complex and difficult to model physically in an accurate manner. The ability to measure impedance of thousands of beads at multiple frequencies, however, allows for a data-driven approach for protein quantification. By moving from a physics driven approach to a data driven approach, we have developed, for the first time ever to the best of our knowledge, an electronic assay using a reusable microfluidic impedance cytometer chip in conjunction with supervised machine learning to quantifying immunoglobulins G (IgG) and immunoglobulins A (IgA) in saliva within two minutes.

Nucleic Acid Quantification by Multi-Frequency Impedance Cytometry and Machine Learning.

Determining nucleic acid concentrations in a sample is an important step prior to proceeding with downstream analysis in molecular diagnostics. Given the need for testing DNA amounts and its purity in many samples, including in samples with very small input DNA, there is utility of novel machine learning approaches for accurate and high-throughput DNA quantification. Here, we demonstrated the ability of a neural network to predict DNA amounts coupled to paramagnetic beads. To this end, a custom-made microfluidic chip is applied to detect DNA molecules bound to beads by measuring the impedance peak response (IPR) at multiple frequencies. We leveraged electrical measurements including the frequency and imaginary and real parts of the peak intensity within a microfluidic channel as the input of deep learning models to predict DNA concentration. Specifically, 10 different deep learning architectures are examined. The results of the proposed regression model indicate that an R_Squared of 97% with a slope of 0.68 is achievable. Consequently, machine learning models can be a suitable, fast, and accurate method to measure nucleic acid concentration in a sample. The results presented in this study demonstrate the ability of the proposed neural network to use the information embedded in raw impedance data to predict the amount of DNA concentration.

Detection of respiratory inflammation biomarkers in non-processed exhaled breath condensate samples using reduced graphene oxide.

In this work, we studied several important parameters regarding the standardization of a portable sensor of nitrite, a key biomarker of inflammation in the respiratory tract in untreated EBC samples. The storage of the EBC samples and electrical properties of both EBC samples and the sensor as main standardization parameters were investigated. The sensor performance was performed using differential pulse voltammetry (DPV) in a standard nitrite solution and untreated EBC samples. The storage effect was monitored by comparing sensor data of fresh and stored samples for one month at -80 °C. Results show, on average, a 20 percent reduction of peak current for stored solutions. The sensor's performance was compared with a previous EBC nitrite sensor and chemiluminescence method. The results demonstrate a good correlation between the present sensor and chemiluminescence for low nitrite concentrations in untreated EBC samples. The electrical behavior of the sensor and electrical variation between EBC samples were characterized using methods such as noise analysis, electrochemical impedance spectroscopy (EIS), electrical impedance (EI), and voltage shift. Data show that reduced graphene oxide (rGO) has lower electrical noise and a higher electron transfer rate regarding nitrite detection. Also, a voltage shift can be applied to calibrate the data based on the electrical variation between different EBC samples. This result makes it easy to calibrate the electrical difference between EBC samples and have a more reproducible portable chip design without using bulky EI instruments. This work helps detect nitrite in untreated and pure EBC samples and evaluates critical analytical EBC properties essential for developing portable and on-site point-of-care sensors.

A portable analog front-end system for label-free sensing of proteins using nanowell array impedance sensors.

Proteins are useful biomarkers for a wide range of applications such as cancer detection, discovery of vaccines, and determining exposure to viruses and pathogens. Here, we present a low-noise front-end analog circuit interface towards development of a portable readout system for the label-free sensing of proteins using Nanowell array impedance sensing with a form factor of approximately 35cm2. The electronic interface consists of a low-noise lock-in amplifier enabling reliable detection of changes in impedance as low as 0.1% and thus detection of proteins down to the picoMolar level. The sensitivity of our system is comparable to that of a commercial bench-top impedance spectroscope when using the same sensors. The aim of this work is to demonstrate the potential of using impedance sensing as a portable, low-cost, and reliable method of detecting proteins, thus inching us closer to a Point-of-Care (POC) personalized health monitoring system. We have demonstrated the utility of our system to detect antibodies at various concentrations and protein (45 pM IL-6) in PBS, however, our system has the capability to be used for assaying various biomarkers including proteins, cytokines, virus molecules and antibodies in a portable setting.

Biosensors and machine learning for enhanced detection, stratification, and classification of cells: a review.

Biological cells, by definition, are the basic units which contain the fundamental molecules of life of which all living things are composed. Understanding how they function and differentiating cells from one another, therefore, is of paramount importance for disease diagnostics as well as therapeutics. Sensors focusing on the detection and stratification of cells have gained popularity as technological advancements have allowed for the miniaturization of various components inching us closer to Point-of-Care (POC) solutions with each passing day. Furthermore, Machine Learning has allowed for enhancement in the analytical capabilities of these various biosensing modalities, especially the challenging task of classification of cells into various categories using a data-driven approach rather than physics-driven. In this review, we provide an account of how Machine Learning has been applied explicitly to sensors that detect and classify cells. We also provide a comparison of how different sensing modalities and algorithms affect the classifier accuracy and the dataset size required.

Development of a portable toolkit to diagnose coral thermal stress.

Coral bleaching, precipitated by the expulsion of the algal symbionts that provide colonies with fixed carbon is a global threat to reef survival. To protect corals from anthropogenic stress, portable tools are needed to detect and diagnose stress syndromes and assess population health prior to extensive bleaching. Here, medical grade Urinalysis strips, used to detect an array of disease markers in humans, were tested on the lab stressed Hawaiian coral species, Montipora capitata (stress resistant) and Pocillopora acuta (stress sensitive), as well as samples from nature that also included Porites compressa. Of the 10 diagnostic reagent tests on these strips, two appear most applicable to corals: ketone and leukocytes. The test strip results from M. capitata were explored using existing transcriptomic data from the same samples and provided evidence of the stress syndromes detected by the strips. We designed a 3D printed smartphone holder and image processing software for field analysis of test strips (TestStripDX) and devised a simple strategy to generate color scores for corals (reflecting extent of bleaching) using a smartphone camera (CoralDX). Our approaches provide field deployable methods, that can be improved in the future (e.g., coral-specific stress test strips) to assess reef health using inexpensive tools and freely available software.

Aluminum Oxide-Coated Particle Differentiation Employing Supervised Machine Learning and Impedance Cytometry.

This article uses a supervised machine learning (ML) system for identifying groups of nanoparticles coated with metal oxides of varying thicknesses using a microfluidic impedance cytometer. These particles generate unique impedance signatures when probed with a multifrequency electric field and finds applications in enabling many multiplexed biosensing technologies. However, current experimental and data processing techniques are unable to sensitively differentiate different metal oxide coated particle types. Here, we employ various machine learning models and collect multiple particle metrics measured. In reported experiments, a 75% accuracy was determined to separate aluminum oxide coated (10nm and 30nm), which is significantly greater than observing only univariate data between different microparticle types. This approach will enable ML models to differentiate such particles with greater accuracies.

Antibody-functionalized aluminum oxide-coated particles targeting neutrophil receptors in a multifrequency microfluidic impedance cytometer.

Personalized diagnostics of infectious diseases require monitoring disease progression due to their ever-changing physiological conditions and the multi-faceted organ system mechanisms involved in disease pathogenesis. In such instances, the recommended clinical strategies involve multiplexing data collection from critical biomarkers related to a patient's conditions along with longitudinal frequent patient monitoring. Numerous detection technologies exist both in research and commercial settings to monitor these conditions, however, they fail to provide biomarker multiplexing ability with design and data processing simplicity. For a recently conceived multiplexing biomarker modality, this work demonstrates the use of electrically sensitive microparticles targeting and identifying membrane receptors on leukocytes using a single detection source, with a high potential for multiplexing greater than any existing impedance-based single-detection scheme. Here, polystyrene microparticles are coated with varying thicknesses of metal oxides, which generate quantifiable impedance shifts when exposed to multifrequency electric fields depending on the metal oxide thickness. Using multifrequency impedance cytometry, these particles can be measured and differentiated rapidly across one coplanar electrode scheme. After surface-functionalizing particles with antibodies targeting CD11b and CD66b receptors, the particles are combined with isolated neutrophils to measure receptor expression. A combination of data analysis techniques including multivariate analysis, supervised machine learning, and unsupervised machine learning was able to accurately differentiate samples with up to 91% accuracy. This proof-of-concept study demonstrates the potential for these oxide-coated particles for enumerating specific leukocytes enabling multiplexing. Further, additional coating thicknesses or different metal oxide materials can enable a compendium of multiplexing targeting resource to be used to develop a high-multiplexing sensor for targeting membrane receptor expression.

A Smartphone-Based Disposable Hemoglobin Sensor Based on Colorimetric Analysis.

Hemoglobin is a biomarker of interest for the diagnosis and prognosis of various diseases such as anemia, sickle cell disease, and thalassemia. In this paper, we present a disposable device that has the potential of being used in a setting for accurately quantifying hemoglobin levels in whole blood based on colorimetric analysis using a smartphone camera. Our biosensor employs a disposable microfluidic chip which is made using medical-grade tapes and filter paper on a glass slide in conjunction with a custom-made PolyDimethylSiloaxane (PDMS) micropump for enhancing capillary flow. Once the blood flows through the device, the glass slide is imaged using a smartphone equipped with a custom 3D printed attachment. The attachment has a Light Emitting Diode (LED) that functions as an independent light source to reduce the noise caused by background illumination and external light sources. We then use the RGB values obtained from the image to quantify the hemoglobin levels. We demonstrated the capability of our device for quantifying hemoglobin in Bovine Hemoglobin Powder, Frozen Beef Blood, and human blood. We present a logarithmic model that specifies the relationship between the Red channel of the RGB values and Hemoglobin concentration.

MACHINE LEARNING ENABLES QUANTIFYING CELL-JANUS PARTICLE CONJUGATES THROUGH MICROFLOWING IMPEDANCE SIGNALS.

In this work, we demonstrate the differentiation of demodulated multifrequency signals from impedance sensitive microparticles when targeting surface receptors on neutrophils in a microfluidic impedance cytometer. These scheme uses a single signal input and detection configuration, and machine learning can differentiate particle types with up to 82% accuracy.

A microwell-based impedance sensor on an insertable microneedle for real-time in vivo cytokine detection.

Impedance-based protein detection sensors for point-of-care diagnostics require quantitative specificity, as well as rapid or real-time operation. Furthermore, microfabrication of these sensors can lead to the formation of factors suitable for in vivo operation. Herein, we present microfabricated needle-shaped microwell impedance sensors for rapid-sample-to-answer, label-free detection of cytokines, and other biomarkers. The microneedle form factor allows sensors to be utilized in transcutaneous or transvascular sensing applications. In vitro, experimental characterization confirmed sensor specificity and sensitivity to multiple proteins of interest. Mechanical characterization demonstrated sufficient microneedle robustness for transcutaneous insertion, as well as preserved sensor function postinsertion. We further utilized these sensors to carry out real-time in vivo quantification of human interleukin 8 (hIL8) concentration levels in the blood of transgenic mice that endogenously express hIL8. To assess sensor functionality, hIL8 concentration levels in serum samples from the same mice were quantified by ELISA. Excellent agreement between real-time in vivo sensor readings in blood and subsequent ELISA serum assays was observed over multiple transgenic mice expressing hIL8 concentrations from 62 pg/mL to 539 ng/mL.

Single-step label-free nanowell immunoassay accurately quantifies serum stress hormones within minutes.

A non-faradaic label-free cortisol sensing platform is presented using a nanowell array design, in which the two probe electrodes are integrated within the nanowell structure. Rapid and low volume (≤5 µl) sensing was realized through functionalizing nanoscale volume wells with antibodies and monitoring the real-time binding events. A 28-well plate biochip was built on a glass substrate by sequential deposition, patterning, and etching steps to create a stack nanowell array sensor with an electrode gap of 40 nm. Sensor response for cortisol concentrations between 1 and 15 µg/dl in buffer solution was recorded, and a limit of detection of 0.5 µg/dl was achieved. Last, 65 human serum samples were collected to compare the response from human serum samples with results from the standard enzyme-linked immunosorbent assay (ELISA). These results confirm that nanowell array sensors could be a promising platform for point-of-care testing, where real-time, laboratory-quality diagnostic results are essential.

Compact and automated particle counting platform using smartphone-microscopy.

The majority of existing advanced automated cell counter instruments used in laboratory settings are complex, expensive, and bulky. As a result, applications of these instruments have been limited to such laboratories. Meanwhile, in many rural areas and developing countries, clinical laboratories equipped with optical microscopy, hematology analyzers or commercial automated particle counters may not be readily accessible to everyone. However, in the same regions, the number of cell-phone users are rapidly increasing, suggesting a need to develop an easy-to-use and portable smart-phone based cell counting technology that can be leveraged in resource-limited areas. To this end, we present an automated, portable and easy-to-use smartphone-based particle counting platform designed to detect particles in a sample solution. This novel pump-less, flow-based portable technology utilizes a small lens attached to a smartphone camera to magnify particles passing through a microfluidic channel, and record a video using a smartphone camera application. The captured video is transmitted to a local computer to be processed through a custom-developed algorithm to perform particle counting. Using a total of 30 different test samples, we have shown that our technology can detect and count polystyrene beads as small as 16.2 µm with ~100% accuracy. We evaluated the performance of our new technology using different size particles and showed that the results are comparable to the tedious, manual-microscope based counting method. Although, we benchmark the performance of the platform using polystyrene beads, we emphasize the wide applicability of the platform to a wide range of biological, biomedical, and industrial applications.