Multiplexed Chemical Sensing CMOS Imager.

A miniaturized and multiplexed chemical sensing technology is urgently needed to empower mobile devices and robots for various new applications such as mobile health and Internet of Things. Here, we show that a complementary metal-oxide-semiconductor (CMOS) imager can be turned into a multiplexed colorimetric sensing chip by coating micron-scale sensing spots on the CMOS imager surface. Each sensing spot contains nanocomposites of colorimetric sensing probes and silica nanoparticles that enhance sensing signals by several orders of magnitude. The sensitivity is spot-size-invariant, and high-performance gas sensing can be achieved on sensing spots as small as ∼10 µm. This great scalability combined with millions of pixels of a CMOS imager offers a promising platform for highly integrated chemical sensors. To prove its compatibility with mobile electronics, we have built a smartphone accessory based on this chemical CMOS sensor and demonstrated that personal health management can be achieved through the detection of gaseous biomarkers and pollutants. We anticipate that this new platform will pave the way for the widespread application of chemical sensing in mobile electronics and wearable devices.

Electrochemically controlled rectification in symmetric single-molecule junctions.

Single-molecule electrochemical science has advanced over the past decades and now extends well beyond molecular imaging, to molecular electronics functions such as rectification and amplification. Rectification is conceptually the simplest but has involved mostly challenging chemical synthesis of asymmetric molecular structures or asymmetric materials and geometry of the two enclosing electrodes. Here we propose an experimental and theoretical strategy for building and tuning in situ (in operando) rectification in two symmetric molecular structures in electrochemical environment. The molecules were designed to conduct electronically via either their lowest unoccupied molecular orbital (LUMO; electron transfer) or highest occupied molecular orbital (HOMO; "hole transfer"). We used a bipotentiostat to control separately the electrochemical potential of the tip and substrate electrodes of an electrochemical scanning tunneling microscope (EC-STM), which leads to independent energy alignment of the STM tip, the molecule, and the STM substrate. By creating an asymmetric energy alignment, we observed single-molecule rectification of each molecule within a voltage range of ±0.5 V. By varying both the dominating charge transporting LUMO or HOMO energy and the electrolyte concentration, we achieved tuning of the polarity as well as the amplitude of the rectification. We have extended an earlier proposed theory that predicts electrolyte-controlled rectification to rationalize all the observed in situ rectification features and found excellent agreement between theory and experiments. Our study thus offers a way toward building controllable single-molecule rectifying devices without involving asymmetric molecular structures.

Rapid Detection of Urinary Tract Infection in 10 min by Tracking Multiple Phenotypic Features in a 30 s Large-Volume Scattering Video of Urine Microscopy.

Rapid point-of-care (POC) diagnosis of bacterial infection diseases provides clinical benefits of prompt initiation of antimicrobial therapy and reduction of the overuse/misuse of unnecessary antibiotics for nonbacterial infections. We present here a POC compatible method for rapid bacterial infection detection in 10 min. We use a large-volume solution scattering imaging (LVSi) system with low magnifications (1-2×) to visualize bacteria in clinical samples, thus eliminating the need for culture-based isolation and enrichment. We tracked multiple intrinsic phenotypic features of individual cells in a short video. By clustering these features with a simple machine learning algorithm, we can differentiate Escherichia coli from similar-sized polystyrene beads, distinguish bacteria with different shapes, and distinguish E. coli from urine particles. We applied the method to detect urinary tract infections in 104 patient urine samples with a 30 s LVSi video, and the results showed 92.3% accuracy compared with the clinical culture results. This technology provides opportunities for rapid bacterial infection diagnosis at POC settings.

Label-Free Quantification of Molecular Interaction in Live Red Blood Cells by Tracking Nanometer Scale Membrane Fluctuations.

Molecular interactions in live cells play an important role in both cellular functions and drug discovery. Current methods for measuring binding kinetics involve extracting the membrane protein and labeling, while the in situ quantification of molecular interaction with surface plasmon resonance (SPR) imaging mainly worked with fixed cells due to the micro-motion related noises of live cells. Here, an optical imaging method is presented to measure the molecular interaction with live red blood cells by tracking the nanometer membrane fluctuations. The membrane fluctuation dynamics are measured by tracking the membrane displacement during glycoprotein interaction. The data are analyzed with a thermodynamic model to determine the elastic properties of the cell observing reduced membrane fluctuations under fixatives, indicating cell fixations affect membrane mechanical properties. The binding kinetics of glycoprotein to several lectins are obtained by tracking the membrane fluctuation amplitude changes on single live cells. The binding kinetics and strength of different lectins are quite different, indicating the glycoproteins expression heterogeneity in single cells. It is anticipated that the method will contribute to the understanding of mechanisms of cell interaction and communication, and have potential applications in the mechanical assessment of cancer or other diseases at the single-cell level, and screening of membrane protein targeting drugs.

Charge Sensitive Optical Detection for Measurement of Small-Molecule Binding Kinetics.

Charge sensitive optical detection (CSOD) technique is a label-free method for real-time measurement of molecular interactions. Traditional label-free optical detection techniques mostly measure the mass of a molecule, and they are less sensitive to small molecules. In contrast, CSOD detects the charge of a molecule, where the signal does not diminish with the size of the molecule, thus capable for studying small molecules. In addition, CSOD is compatible with the standard microplate platform, making it suitable for high-throughput screening of drug candidates. In CSOD, an optical fiber functionalized with the probe molecule is dipped into a well of a microplate where an alternate perpendicular electrical field is applied to the fiber, which drives the fiber into oscillation because of the presence of surface charge on the fiber. The binding of the target molecules changes the charge of the fiber, and thus the amplitude and phase of the oscillating fiber, which are precisely measured through tracking of the optical images of the fiber tip.

Single-molecule calorimeter and free energy landscape.

The precise measurement of thermodynamic and kinetic properties for biomolecules provides the detailed information for a multitude of applications in biochemistry, biosensing, and health care. However, sensitivity in characterizing the thermodynamic binding affinity down to a single molecule, such as the Gibbs free energy ([Formula: see text]), enthalpy ([Formula: see text]), and entropy ([Formula: see text]), has not materialized. Here, we develop a nanoparticle-based technique to probe the energetic contributions of single-molecule binding events, which introduces a focused laser of optical tweezer to an optical path of plasmonic imaging to accumulate and monitor the transient local heating. This single-molecule calorimeter uncovers the complex nature of molecular interactions and binding characterizations, which can be employed to identify the thermodynamic equilibrium state and determine the energetic components and complete thermodynamic profile of the free energy landscape. This sensing platform promises a breakthrough in measuring thermal effect at the single-molecule level and provides a thorough description of biomolecular specific interactions.

Rapid Antimicrobial Susceptibility Testing on Clinical Urine Samples by Video-Based Object Scattering Intensity Detection.

To combat the ongoing public health threat of antibiotic-resistant infections, a technology that can quickly identify infecting bacterial pathogens and concurrently perform antimicrobial susceptibility testing (AST) in point-of-care settings is needed. Here, we develop a technology for point-of-care AST with a low-magnification solution scattering imaging system and a real-time video-based object scattering intensity detection method. The low magnification (1-2×) optics provides sufficient volume for direct imaging of bacteria in urine samples, avoiding the time-consuming process of culture-based bacterial isolation and enrichment. Scattering intensity from moving bacteria and particles in the sample is obtained by subtracting both spatial and temporal background from a short video. The time profile of scattering intensity is correlated with the bacterial growth rate and bacterial response to antibiotic exposure. Compared to the image-based bacterial tracking and counting method we previously developed, this simple image processing algorithm accommodates a wider range of bacterial concentrations, simplifies sample preparation, and greatly reduces the computational cost of signal processing. Furthermore, development of this simplified processing algorithm eases implementation of multiplexed detection and allows real-time signal readout, which are essential for point-of-care AST applications. To establish the method, 130 clinical urine samples were tested, and the results demonstrated an accuracy of ∼92% within 60-90 min for UTI diagnosis. Rapid AST of 55 positive clinical samples revealed 98% categorical agreement with both the clinical culture results and the on-site parallel AST validation results. This technology provides opportunities for prompt infection diagnosis and accurate antibiotic prescriptions in point-of-care settings.

Colorimetric Sensor for Online Accurate Detection of Breath Acetone.

Breath acetone (BrAce) is a validated biomarker of lipid oxidation and has been extensively studied for many applications, such as monitoring ketoacidosis in diabetes, guiding ketogenic diet, and measuring fat burning during exercise. Although many sensors have been reported for BrAce measurement, most of the contributions tested only synthetic or spiked breath samples, because of the complex components of human breath. Here, we show that online accurate detection of BrAce can be achieved using a colorimetric sensor. The high selectivity is enabled by the specific reaction between acetone and hydroxylamine sulfate, and the sensor has a high agreement with a reference instrument in ketosis monitoring. We anticipate that the colorimetric acetone sensor can be applied to various health-related applications.

Plasmonic Imaging of Oxidation and Reduction of Single Gold Nanoparticles and Their Surface Structural Dynamics.

Gold nanoparticles (AuNPs) have been widely used in catalytic electrochemistry. Heterogeneity in size, shape, and surface sites leads to variable, particle-specific catalytic activities. Conventional electrochemical methods can only obtain the collective responses from all the catalytic nanoparticles on the electrode surface; the heterogeneity of particle performance will be averaged. Alternatively, plasmonic electrochemical imaging (PECi) is capable of imaging the electrochemical activity at individual nanoparticles. In this work, PECi was used to image the oxidation and reduction of the gold surface at individual AuNPs, and their associated structural alterations were successfully measured. We have studied the electrochemical responses from gold nanocubes, gold nanorods, and gold nanowires with PECi and observed different surface redox activities. We have also demonstrated the capability of monitoring the surface dynamics at individual AuNPs utilizing characteristic PECi derived cyclic voltammograms (CVs).

Gradient-Based Rapid Digital Immunoassay for High-Sensitivity Cardiac Troponin T (hs-cTnT) Detection in 1 µL Plasma.

Rapid and sensitive detection of biomarkers is the key to the diagnosis of acute diseases. One example is the detection of troponin in myocardial infarction. Here, we report a gradient-based digital immunoassay method, which can achieve high-sensitivity cardiac troponin T (hs-cTnT) detection with only 1 µL of plasma sample. We designed a multizone microfluidic channel functionalized with capture antibody specific to troponin. Taking advantage of limited sample volume, a troponin concentration gradient is created along the channel because of binding induced depletion. We quantified the concentration gradient by counting the detection antibody conjugated gold nanoparticles bound to different test zones with optical imaging. Differential counting between the zones removes most common noises and nonspecific bindings. The total analytical time is about 30 min, and the limit of quantification is 6.2 ng/L. We examined 41 clinical plasma samples from 15 patients and the change in hs-cTnT concentration in serial samples showed good linear correlation with clinical results (R2 = 0.98). Therefore, this simple and sensitive gradient-based digital immunoassay method is a promising technology for clinical hs-cTnT detection and could be adapted for detection of other biomarkers.

Imaging Single Bacterial Cells with Electro-optical Impedance Microscopy.

Impedance measurements have been an important tool for biosensor applications, including protein detection, DNA quantification, and cell study. We present here an electro-optical impedance microscopy (EIM) based on the dependence of surface optical transmission on local surface charge density for single bacteria impedance imaging. We applied a potential modulation to bacteria placed on an indium tin oxide-coated slide and simultaneously recorded a sequence of transmitted microscopy images. By performing fast Fourier transform analysis on the image sequences, we obtained the DC component (signal at 0 Hz) for cell morphology imaging and the AC component (signal at the modulation frequency) for the mapping of cell impedance responses with subcellular resolution for the first time. Using this method, we have monitored the viability of Escherichia coli bacterial cells under treatment with two different classes of antibiotics with low-frequency potential modulation. The results showed that the impedance response is sensitive to the antibiotic that targets the bacterial cell membrane as the membrane capacitance dominated at low-frequency modulation. Heterogeneous responses to the antibiotic treatment were observed at a single bacteria level. In addition to the high spatial resolution, EIM is label-free and simple and can be potentially used for the continuous mapping of single bacteria impedance changes under different conditions.

Charge-Sensitive Optical Detection of Small Molecule Binding Kinetics in Normal Ionic Strength Buffer.

Most label-free detection technologies detect the masses of molecules, and their sensitivities thus decrease with molecular weight, making it challenging to detect small molecules. To address this need, we have developed a charge-sensitive optical detection (CSOD) technique, which detects the charge rather than the mass of a molecule with an optical fiber. However, the effective charge of a molecule decreases with the buffer ionic strength. For this reason, the previous CSOD works with diluted buffers, which could affect the measured molecular binding kinetics. Here, we show a technique capable of detecting molecular binding kinetics in normal ionic strength buffers. An H-shaped sample well was developed to increase the current density at the sensing area to compensate the signal loss due to ionic screening at normal ionic strength buffer, while keeping the current density low at the electrodes to minimize the electrode reaction. In addition, agarose gels were used to cover the electrodes to prevent electrode reaction generated bubbles from entering the sensing area. With this new design, we have measured the binding kinetics between G-protein-coupled receptors (GPCRs) and their small molecule ligands in normal buffer. We found that the affinities measured in normal buffer are stronger than those measured in diluted buffer, likely due to the stronger electrostatic repulsion force between the same charged ligands and receptors in the diluted buffer.

Probing Single-Molecule Binding Event by the Dynamic Counting and Mapping of Individual Nanoparticles.

Measuring binding processes at the single-molecule level underpin significant functions in understanding biological events. Single-nanoparticle imaging techniques are providing a new concept for mapping the heterogeneous behaviors and characterizations of individual dynamics such as molecule-molecule interactions. Here, we develop the optical imaging techniques for directly counting and monitoring the binding and motion events of single nanoparticles linked to the substrate via the specific and reversible interactions between biomolecules. The one-step digital immunoassay realizes the biomolecular detection based on dynamic counting of the single nanoparticle binding event to substrate with the bright-field imaging. The detection limit achieves 8.4 pg/mL for procalcitonin with detection time of 14 min. Meanwhile, we map the accurate trajectory of single nanoparticle switching between different target molecules among the x-y plane with the total internal reflection imaging technique, which reveals the spatial coordinates of single target molecules on the substrate surface with high spatial and temporal resolutions.

Gradient-Based Colorimetric Array Sensor for Continuous Monitoring of Multiple Gas Analytes.

Colorimetry is widely used in chemical sensing due to its high sensitivity and high selectivity. However, most colorimetric sensors are one-time use because the color-producing reactions or bindings are usually irreversible. In addition, traditional colorimetric sensors like the detection tubes are bulky and packed individually, making parallel sensing of multiple analytes difficult. Here, we demonstrate a gradient-based colorimetric array sensor (GCAS) to overcome these limitations. Different colorimetric sensing elements are inkjet-printed as parallel straight lines on a porous substrate. Lateral transport of analytes across the substrate creates color gradients on the sensing elements. The color gradients shift along the transport direction over time, and GCAS tracks the gradient shifts and converts them into analyte concentrations in real time. Using a low-cost complementary metal-oxide semiconductor imager, we show detection of three air pollutants using a single GCAS chip and 24 h continuous monitoring of ambient ozone.

Optical Imaging of Electrical and Mechanical Couplings between Cells.

Intercellular communication plays a pivotal role in multicellular organisms. Studying the electrical and mechanical coupling among multiple cells has been a difficult task due to the lack of suitable techniques. In this study, we developed a label-free imaging method for monitoring the electrical-induced communications between connected cells. The method was based on monitoring subtle mechanical motions of the cells under electrical modulation of the membrane potential. We observed that connected cells responded to electrical modulation of neighboring cells with mechanical deformation of the membrane. We further investigated the mechanism of the coupling and confirmed that this mechanical response was induced by electrical signal communicated through the gap junction. Blocking the gap junction can temporally cease the mechanical signal, and this inhibition can be rescued after removing the inhibitor. This study sheds light on the mechanism of electrical coupling between neurons and provides a new method for studying intercellular communications.

Roles of entropic and solvent damping forces in the dynamics of polymer tethered nanoparticles and implications for single molecule sensing.

Tethering a particle to a surface with a single molecule allows detection of the molecule and analysis of molecular conformations and interactions. Understanding the dynamics of the system is critical to all applications. Here we present a plasmonic imaging study of two important forces that govern the dynamics. One is entropic force arising from the conformational change of the molecular tether, and the other is solvent damping on the particle and the molecule. We measure the response of the particle by driving it into oscillation with an alternating electric field. By varying the field frequency, we study the dynamics on different time scales. We also vary the type of the tether molecule (DNA and polyethylene glycol), size of the particle, and viscosity of the solvent, and describe the observations with a model. The study allows us to derive a single parameter to predict the relative importance of the entropic and damping forces. The findings provide insights into single molecule studies using not only tethered particles, but also other approaches, including force spectroscopy using atomic force microscopy and nanopores.

An Unobstructive Sensing Method for Indoor Air Quality Optimization and Metabolic Assessment within Vehicles.

This work investigates the use of an intelligent and unobstructive sensing technique for maintaining vehicle cabin's indoor air quality while simultaneously assessing the driver metabolic rate. CO2 accumulation patterns are of great interest because CO2 can have negative cognitive effects at higher concentrations and also since CO2 accumulation rate can potentially be used to determine a person's metabolic rate. The management of the vehicle's ventilation system was controlled by periodically alternating the air recirculation mode within the cabin, which was actuated based on the CO2 levels inside the vehicle's cabin. The CO2 accumulation periods were used to assess the driver's metabolic rate, using a model that considered the vehicle's air exchange rate. In the process of the method optimization, it was found that the vehicle's air exchange rate (λ [h-1]) depends on the vehicle speeds, following the relationship: λ = 0.060 × (speed) - 0.88 when driving faster than 17 MPH. An accuracy level of 95% was found between the new method to assess the driver's metabolic rate (1620 ± 140 kcal/day) and the reference method of indirect calorimetry (1550 ± 150 kcal/day) for a total of N = 16 metabolic assessments at various vehicle speeds. The new sensing method represents a novel approach for unobstructive assessment of driver metabolic rate while maintaining indoor air quality within the vehicle cabin.

Direct Antimicrobial Susceptibility Testing on Clinical Urine Samples by Optical Tracking of Single Cell Division Events.

With the increasing prevalence of antibiotic resistance, the need to develop antimicrobial susceptibility testing (AST) technologies is urgent. The current challenge has been to perform the antibiotic susceptibility testing in short time, directly with clinical samples, and with antibiotics over a broad dynamic range of clinically relevant concentrations. Here, a technology for point-of-care diagnosis of antimicrobial-resistant bacteria in urinary tract infections, by imaging the clinical urine samples directly with an innovative large volume solution scattering imaging (LVSi) system and analyzing the image sequences with a single-cell division tracking method is developed. The high sensitivity of single-cell division tracking associated with large volume imaging enables rapid antibiotic susceptibility testing directly on the clinical urine samples. The results demonstrate direct detection of bacterial infections in 60 clinical urine samples with a 60 min LVSi video, and digital AST of 30 positive clinical samples with 100% categorical agreement with both the clinical culture results and the on-site agar plating validation results. This technology provides opportunities for precise antibiotic prescription and proper treatment of the patient within a single clinic visit.

Conductance and configuration of molecular gold-water-gold junctions under electric fields.

Water molecules can mediate charge transfer in biological and chemical reactions by forming electronic coupling pathways. Understanding the mechanism requires a molecular-level electrical characterization of water. Here, we describe the measurement of single water molecular conductance at room temperature, characterize the structure of water molecules using infrared spectroscopy, and perform theoretical studies to assist in the interpretation of the experimental data. The study reveals two distinct states of water, corresponding to a parallel and perpendicular orientation of the molecules. Water molecules switch from parallel to perpendicular orientations on applying an electric field, producing switching from high to low conductance states, thus enabling the determination of single water molecular dipole moments. The work further shows that water-water interactions affect the atomic scale configuration and conductance of water molecules. These findings demonstrate the importance of the discrete nature of water molecules in electron transfer and set limits on water-mediated electron transfer rates.

Optical imaging of single-protein size, charge, mobility, and binding.

Detection and identification of proteins are typically achieved by analyzing protein size, charge, mobility and binding to antibodies, which are critical for biomedical research and disease diagnosis and treatment. Despite the importance, measuring these quantities with one technology and at the single-molecule level has not been possible. Here we tether a protein to a surface with a flexible polymer, drive it into oscillation with an electric field, and image the oscillation with a near field optical imaging method, from which we determine the size, charge, and mobility of the protein. We also measure antibody binding and conformation changes in the protein. The work demonstrates a capability for comprehensive protein analysis and precision protein biomarker detection at the single molecule level.