Skin-integrated systems for power efficient, programmable thermal sensations across large body areas.

Thermal sensations contribute to our ability to perceive and explore the physical world. Reproducing these sensations in a spatiotemporally programmable manner through wireless computer control could enhance virtual experiences beyond those supported by video, audio and, increasingly, haptic inputs. Flexible, lightweight and thin devices that deliver patterns of thermal stimulation across large areas of the skin at any location of the body are of great interest in this context. Applications range from those in gaming and remote socioemotional communications, to medical therapies and physical rehabilitation. Here, we present a set of ideas that form the foundations of a skin-integrated technology for power-efficient generation of thermal sensations across the skin, with real-time, closed-loop control. The systems exploit passive cooling mechanisms, actively switchable thermal barrier interfaces, thin resistive heaters and flexible electronics configured in a pixelated layout with wireless interfaces to portable devices, the internet and cloud data infrastructure. Systematic experimental studies and simulation results explore the essential mechanisms and guide the selection of optimized choices in design. Demonstration examples with human subjects feature active thermoregulation, virtual social interactions, and sensory expansion.

A Sewing Approach to the Fabrication of Eco/bioresorbable Electronics.

Eco/bioresorbable electronics represent an emerging class of technology defined by an ability to dissolve or otherwise harmlessly disappear in environmental or biological surroundings after a period of stable operation. The resulting devices provide unique capabilities as temporary biomedical implants, environmental sensors, and related systems. Recent publications report schemes to overcome challenges in fabrication that follow from the low thermostability and/or high chemical reactivity of the eco/bioresorbable constituent materials. Here, this work reports the use of high-speed sewing machines, as the basis for a high-throughput manufacturing technique that addresses many requirements for these applications, without the need for high temperatures or reactive solvents. Results demonstrate that a range of eco/bioresorbable metal wires and polymer threads can be embroidered into complex, user-defined conductive patterns on eco/bioresorbable substrates. Functional electronic components, such as stretchable interconnects and antennas are possible, along with fully integrated systems. Examples of the latter include wirelessly powered light-emitting diodes, radiofrequency identification tags, and temporary cardiac pacemakers. These advances add to a growing range of options in high-throughput, automated fabrication of eco/bioresorbable electronics.

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.

Smartphone clip-on instrument and microfluidic processor for rapid sample-to-answer detection of Zika virus in whole blood using spatial RT-LAMP.

Rapid, simple, inexpensive, accurate, and sensitive point-of-care (POC) detection of viral pathogens in bodily fluids is a vital component of controlling the spread of infectious diseases. The predominant laboratory-based methods for sample processing and nucleic acid detection face limitations that prevent them from gaining wide adoption for POC applications in low-resource settings and self-testing scenarios. Here, we report the design and characterization of an integrated system for rapid sample-to-answer detection of a viral pathogen in a droplet of whole blood comprised of a 2-stage microfluidic cartridge for sample processing and nucleic acid amplification, and a clip-on detection instrument that interfaces with the image sensor of a smartphone. The cartridge is designed to release viral RNA from Zika virus in whole blood using chemical lysis, followed by mixing with the assay buffer for performing reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) reactions in six parallel microfluidic compartments. The battery-powered handheld detection instrument uniformly heats the compartments from below, and an array of LEDs illuminates from above, while the generation of fluorescent reporters in the compartments is kinetically monitored by collecting a series of smartphone images. We characterize the assay time and detection limits for detecting Zika RNA and gamma ray-deactivated Zika virus spiked into buffer and whole blood and compare the performance of the same assay when conducted in conventional PCR tubes. Our approach for kinetic monitoring of the fluorescence-generating process in the microfluidic compartments enables spatial analysis of early fluorescent "bloom" events for positive samples, in an approach called "Spatial LAMP" (S-LAMP). We show that S-LAMP image analysis reduces the time required to designate an assay as a positive test, compared to conventional analysis of the average fluorescent intensity of the entire compartment. S-LAMP enables the RT-LAMP process to be as short as 22 minutes, resulting in a total sample-to-answer time in the range of 17-32 minutes to distinguish positive from negative samples, while demonstrating a viral RNA detection as low as 2.70 × 102 copies per µl, and a gamma-irradiated virus of 103 virus particles in a single 12.5 µl droplet blood sample.

Overcoming the limitations of COVID-19 diagnostics with nanostructures, nucleic acid engineering, and additive manufacturing.

The COVID-19 pandemic revealed fundamental limitations in the current model for infectious disease diagnosis and serology, based upon complex assay workflows, laboratory-based instrumentation, and expensive materials for managing samples and reagents. The lengthy time delays required to obtain test results, the high cost of gold-standard PCR tests, and poor sensitivity of rapid point-of-care tests contributed directly to society's inability to efficiently identify COVID-19-positive individuals for quarantine, which in turn continues to impact return to normal activities throughout the economy. Over the past year, enormous resources have been invested to develop more effective rapid tests and laboratory tests with greater throughput, yet the vast majority of engineering and chemistry approaches are merely incremental improvements to existing methods for nucleic acid amplification, lateral flow test strips, and enzymatic amplification assays for protein-based biomarkers. Meanwhile, widespread commercial availability of new test kits continues to be hampered by the cost and time required to develop single-use disposable microfluidic plastic cartridges manufactured by injection molding. Through development of novel technologies for sensitive, selective, rapid, and robust viral detection and more efficient approaches for scalable manufacturing of microfluidic devices, we can be much better prepared for future management of infectious pathogen outbreaks. Here, we describe how photonic metamaterials, graphene nanomaterials, designer DNA nanostructures, and polymers amenable to scalable additive manufacturing are being applied towards overcoming the fundamental limitations of currently dominant COVID-19 diagnostic approaches. In this paper, we review how several distinct classes of nanomaterials and nanochemistry enable simple assay workflows, high sensitivity, inexpensive instrumentation, point-of-care sample-to-answer virus diagnosis, and rapidly scaled manufacturing.

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.

Comparison of ISO work of breathing and NIOSH breathing resistance measurements for air-purifying respirators.

The National Institute for Occupational Safety and Health's methods and requirements for air-purifying respirator breathing resistance in 42 CFR Part 84 do not include work of breathing. The International Organization for Standardization Technical Committee 94, Subcommittee 15 utilized work of breathing to evaluate airflow resistance for all classes of respiratory protective devices as part of their development of performance standards regarding respiratory protective devices. The objectives of this study were: (1) to evaluate the relationship between the International Organization for Standardization's work of breathing measurements and the National Institute for Occupational Safety and Health's breathing resistance test results; (2) to provide scientific bases for standard development organizations to decide if work of breathing should be adopted; and (3) to establish regression equations for manufacturers and test laboratories to estimate work of breathing measurements using breathing resistance data. A total of 43 respirators were tested for work of breathing at minute ventilation rates of 10, 35, 65, 105, and 135 liters per minute. Breathing resistance obtained at a constant flow rate of 85 liters per minute per National Institute of Occupational Safety and Health protocol was correlated to each of the parameters (total work of breathing, inhalation, and exhalation) obtained from the work of breathing tests. The ratio of work of breathing exhalation to work of breathing inhalation for all air-purifying respirators is similar to the ratio of exhalation to inhalation resistance when tested individually. The ratios were about 0.8 for filtering facepiece respirators, 0.5 for half-masks, and 0.25 for full-facepiece respirators. The National Institute for Occupational Safety and Health's breathing resistance is close to work of breathing's minute ventilation of 35 liters per minute, which represents the common walking/working pace in most workplaces. The work of breathing and the National Institute of Occupational Safety and Health's breathing resistance were found to be strongly and positively correlated (r values of 0.7-0.9) at each work rate for inhalation and exhalation. In addition, linear and multiple regression models (R-squared values of 0.5-0.8) were also established to estimate work of breathing using breathing resistance. Work of breathing was correlated higher to breathing resistance for full-facepiece and half-mask elastomeric respirators than filtering facepiece respirators for inhalation. For exhalation, filtering facepiece respirators were correlated much better than full-facepiece and half-mask elastomeric respirators. Therefore, the National Institute for Occupational Safety and Health's breathing resistance may reasonably be used to predict work of breathing for air-purifying respirators. The results could also be used by manufacturers for product development and evaluation.

Monolayer MoS2 Nanoribbon Transistors Fabricated by Scanning Probe Lithography.

Monolayer MoS2 is a promising material for nanoelectronics; however, the lack of nanofabrication tools and processes has made it very challenging to realize nanometer-scale electronic devices from monolayer MoS2. Here, we demonstrate the fabrication of monolayer MoS2 nanoribbon field-effect transistors as narrow as 30 nm using scanning probe lithography (SPL). The SPL process uses a heated nanometer-scale tip to deposit narrow nanoribbon polymer structures onto monolayer MoS2. The polymer serves as an etch mask during a XeF2 vapor etch, which defines the channel of a field-effect transistor (FET). We fabricated seven devices with a channel width ranging from 30 to 370 nm, and the fabrication process was carefully studied by electronic measurements made at each process step. The nanoribbon devices have a current on/off ratio > 104 and an extrinsic field-effect mobility up to 8.53 cm2/(V s). By comparing a 30 nm wide device with a 60 nm wide device that was fabricated on the same MoS2 flake, we found the narrower device had a smaller mobility, a lower on/off ratio, and a larger subthreshold swing. To our knowledge, this is the first published work that describes a working transistor device from monolayer MoS2 with a channel width smaller than 100 nm.

Holographic patterning of high-performance on-chip 3D lithium-ion microbatteries.

As sensors, wireless communication devices, personal health monitoring systems, and autonomous microelectromechanical systems (MEMS) become distributed and smaller, there is an increasing demand for miniaturized integrated power sources. Although thin-film batteries are well-suited for on-chip integration, their energy and power per unit area are limited. Three-dimensional electrode designs have potential to offer much greater power and energy per unit area; however, efforts to date to realize 3D microbatteries have led to prototypes with solid electrodes (and therefore low power) or mesostructured electrodes not compatible with manufacturing or on-chip integration. Here, we demonstrate an on-chip compatible method to fabricate high energy density (6.5 µWh cm(-2)⋅µm(-1)) 3D mesostructured Li-ion microbatteries based on LiMnO2 cathodes, and NiSn anodes that possess supercapacitor-like power (3,600 µW cm(-2)⋅µm(-1) peak). The mesostructured electrodes are fabricated by combining 3D holographic lithography with conventional photolithography, enabling deterministic control of both the internal electrode mesostructure and the spatial distribution of the electrodes on the substrate. The resultant full cells exhibit impressive performances, for example a conventional light-emitting diode (LED) is driven with a 500-µA peak current (600-C discharge) from a 10-µm-thick microbattery with an area of 4 mm(2) for 200 cycles with only 12% capacity fade. A combined experimental and modeling study where the structural parameters of the battery are modulated illustrates the unique design flexibility enabled by 3D holographic lithography and provides guidance for optimization for a given application.

Evidence of differential mass change rates between human breast cancer cell lines in culture.

Investigating the growth signatures of single cells will determine how cell growth is regulated and cell size is maintained. The ability to precisely measure such changes and alterations in cell size and cell mass could be important for applications in cancer and drug screening. Here, we measure the mass growth rate of individual benign (MCF-10A), non-invasive (MCF-7), and highly-invasive malignant (MDA-MB-231) breast cancer cells. A micro-patterning technique was employed to allow for the long-term growth of motile cells. Results show mass growth rates at 4.8%, 1.2%, and 2.8% for MCF-10A, MCF-7, and MDA-MB-231, demonstrating that normal cells have a higher mass growth rate than cancerous cells. All the cell lines show an increase in mass change rate indicating that the mass accumulation rate is exponential over a single cell cycle. The growth rates measured with our MEMS sensor are compared with doubling times obtained through conventional bulk analysis techniques, and exhibit excellent agreement.

Micromechanical contact stiffness devices and application for calibrating contact resonance atomic force microscopy.

This paper reports the design, fabrication, and characterization of micromechanical devices that can present an engineered contact stiffness to an atomic force microscope (AFM) cantilever tip. These devices allow the contact stiffness between the AFM tip and a substrate to be easily and accurately measured, and can be used to calibrate the cantilever for subsequent mechanical property measurements. The contact stiffness devices are rigid copper disks of diameters 2-18 µm integrated onto a soft silicone substrate. Analytical modeling and finite element simulations predict the elastic response of the devices. Measurements of tip-sample interactions during quasi-static force measurements compare well with modeling simulation, confirming the expected elastic response of the devices, which are shown to have contact stiffness 32-156 N m-1. To demonstrate one application, we use the disk sample to calibrate three resonant modes of a U-shaped AFM cantilever actuated via Lorentz force, at approximately 220, 450, and 1200 kHz. We then use the calibrated cantilever to determine the contact stiffness and elastic modulus of three polymer samples at these modes. The overall approach allows cantilever calibration without prior knowledge of the cantilever geometry or its resonance modes, and could be broadly applied to both static and dynamic measurements that require AFM calibration against a known contact stiffness.

Three-Dimensional Integration of Graphene via Swelling, Shrinking, and Adaptation.

The transfer of graphene from its growth substrate to a target substrate has been widely investigated for its decisive role in subsequent device integration and performance. Thus far, various reported methods of graphene transfer have been mostly limited to planar or curvilinear surfaces due to the challenges associated with fractures from local stress during transfer onto three-dimensional (3D) microstructured surfaces. Here, we report a robust approach to integrate graphene onto 3D microstructured surfaces while maintaining the structural integrity of graphene, where the out-of-plane dimensions of the 3D features vary from 3.5 to 50 µm. We utilized three sequential steps: (1) substrate swelling, (2) shrinking, and (3) adaptation, in order to achieve damage-free, large area integration of graphene on 3D microstructures. Detailed scanning electron microscopy, atomic force microscopy, Raman spectroscopy, and electrical resistance measurement studies show that the amount of substrate swelling as well as the flexural rigidities of the transfer film affect the integration yield and quality of the integrated graphene. We also demonstrate the versatility of our approach by extension to a variety of 3D microstructured geometries. Lastly, we show the integration of hybrid structures of graphene decorated with gold nanoparticles onto 3D microstructure substrates, demonstrating the compatibility of our integration method with other hybrid nanomaterials. We believe that the versatile, damage-free integration method based on swelling, shrinking, and adaptation will pave the way for 3D integration of two-dimensional (2D) materials and expand potential applications of graphene and 2D materials in the future.

Measuring physical properties of neuronal and glial cells with resonant microsensors.

Microelectromechanical systems (MEMS) resonant sensors provide a high degree of accuracy for measuring the physical properties of chemical and biological samples. These sensors enable the investigation of cellular mass and growth, though previous sensor designs have been limited to the study of homogeneous cell populations. Population heterogeneity, as is generally encountered in primary cultures, reduces measurement yield and limits the efficacy of sensor mass measurements. This paper presents a MEMS resonant pedestal sensor array fabricated over through-wafer pores compatible with vertical flow fields to increase measurement versatility (e.g., fluidic manipulation and throughput) and allow for the measurement of heterogeneous cell populations. Overall, the improved sensor increases capture by 100% at a flow rate of 2 µL/min, as characterized through microbead experiments, while maintaining measurement accuracy. Cell mass measurements of primary mouse hippocampal neurons in vitro, in the range of 0.1-0.9 ng, demonstrate the ability to investigate neuronal mass and changes in mass over time. Using an independent measurement of cell volume, we find cell density to be approximately 1.15 g/mL.

Speed dependence of thermochemical nanolithography for gray-scale patterning.

Thermochemical nanolithography (TCNL) is a high-resolution lithographic technique and, owing to its fast speed, versatility, and unique ability to fabricate arbitrary, gray-scale nanopatterns, this scanning probe technique is relevant both for fundamental scientific research as well as for nanomanufacturing applications. In this work, we study the dependence of the TCNL driven chemical reactions on the translation speed of the thermal cantilever. The experimental data compares well with a model of the chemical kinetics for a first-order reaction. The impact of higher order reactions on the optimization of TCNL is addressed. The reported quantitative description of the speed dependence of TCNL is exploited and illustrated by fabricating controlled gradients of chemical concentration.

Single nanoparticle detection using photonic crystal enhanced microscopy.

We demonstrate a label-free biosensor imaging approach that utilizes a photonic crystal (PC) surface to detect surface attachment of individual dielectric and metal nanoparticles through measurement of localized shifts in the resonant wavelength and resonant reflection magnitude from the PC. Using a microscopy-based approach to scan the PC resonant reflection properties with 0.6 µm spatial resolution, we show that metal nanoparticles attached to the biosensor surface with strong absorption at the resonant wavelength induce a highly localized reduction in reflection efficiency and are able to be detected by modulation of the resonant wavelength. Experimental demonstrations of single-nanoparticle imaging are supported by finite-difference time-domain computer simulations. The ability to image surface-adsorption of individual nanoparticles offers a route to single molecule biosensing, in which the particles can be functionalized with specific recognition molecules and utilized as tags.

An investigation of heat transfer between a microcantilever and a substrate for improved thermal topography imaging.

This paper reports the numerical and experimental investigation of heat transfer from a heated microcantilever to a substrate and uses the resulting insights to improve thermal topography imaging. The cantilever sensitivity, defined as change in thermal signal due to changes in the topography height, is relatively constant for feature heights in the range 100-350 nm. Since the cantilever-substrate heat transfer is governed by thermal conduction through the air, the cantilever sensitivity is nearly constant across substrates of varying thermal conductivity. Surface features with lateral size larger than 2.5 µm can induce artifacts in the cantilever signal resulting in measurement errors as large as 28%. These artifacts arise from thermal conduction from the cantilever in the lateral direction, parallel to the surface. We show how these artifacts can be removed by accounting for this lateral conduction and removing it from the thermal signal. This technique reduces the measurement error by as much as 26%, can be applied to arbitrary substrate topographies, and can be scaled to arrays of heated cantilevers. These results could lead to improvements in nanometer-scale thermal measurements including scanning thermal microscopy and tip-based nanofabrication.

Parallel nanoimaging and nanolithography using a heated microcantilever array.

We report parallel topographic imaging and nanolithography using heated microcantilever arrays integrated into a commercial atomic force microscope (AFM). The array has five AFM cantilevers, each of which has an internal resistive heater. The temperatures of the cantilever heaters can be monitored and controlled independently and in parallel. We perform parallel AFM imaging of a region of size 550 µm × 90 µm, where the cantilever heat flow signals provide a measure of the nanometer-scale substrate topography. At a cantilever scan speed of 1134 µm s(-1), we acquire a 3.1 million-pixel image in 62 s with noise-limited vertical resolution of 0.6 nm and pixels of size 351 nm × 45 nm. At a scan speed of 4030 µm s(-1) we acquire a 26.4 million pixel image in 124 s with vertical resolution of 5.4 nm and pixels of size 44 nm × 43 nm. Finally, we demonstrate parallel nanolithography with the cantilever array, including iterations of measure-write-measure nanofabrication, with each cantilever operating independently.

Silicon nano-mechanical resonators fabricated by using tip-based nanofabrication.

We report fabrication of silicon nano-mechanical resonators where the key nanolithography step is performed by using tip-based nanofabrication (TBN). Specifically, a heated atomic force microscope tip deposited polystyrene nanowires that were used together with a lithographically patterned aluminum to serve as an etch mask for silicon resonators their anchors. Using this nanofabrication technique, we demonstrate the fabrication of different types of silicon nano-mechanical resonator devices, including those that are either singly or doubly clamped and having either straight or curvilinear features. Typical dimensions for the width and thickness of these devices is in the range of several hundred nanometers. We characterized the mechanical resonance properties of these devices by using laser Doppler vibrometry and compared the measured response with finite element simulations. Typical resonance frequency values ranged from 1 to 3 MHz and typical quality factor values ranged from 100 to 150. The combination of TBN along with conventional microfabrication processes could help to realize new types of nano-devices.

Nanofluidic channels of arbitrary shapes fabricated by tip-based nanofabrication.

Nanofluidic channels have promising applications in biomolecule manipulation and sensing. While several different methods of fabrication have been demonstrated for nanofluidic channels, a rapid, low-cost fabrication method that can fabricate arbitrary shapes of nanofluidic channels is still in demand. Here, we report a tip-based nanofabrication (TBN) method for fabricating nanofluidic channels using a heated atomic force microscopy (AFM) tip. The heated AFM tip deposits polymer nanowires where needed to serve as etch mask to fabricate silicon molds through one step of etching. PDMS nanofluidic channels are easily fabricated through replicate molding using the silicon molds. Various shapes of nanofluidic channels with either straight or curvilinear features are demonstrated. The width of the nanofluidic channels is 500 nm, and is determined by the deposited polymer nanowire width. The height of the channel is 400 nm determined by the silicon etching time. Ion conductance measurement on one single curvy shaped nanofluidic channel exhibits the typical ion conductance saturation phenomenon as the ion concentration decreases. Moreover, fluorescence imaging of fluid flowing through a fabricated nanofluidic channel demonstrates the channel integrity. This TBN process is seamlessly compatible with existing nanofabrication processes and can be used to achieve new types of nanofluidic devices.

Multifunctional atomic force microscope cantilevers with Lorentz force actuation and self-heating capability.

This paper reports the development of microcantilevers capable of self-heating and Lorentz-force actuation, and demonstrates applications to thermal topography imaging. Electrical current passing through a U-shaped cantilever in the presence of a magnetic field induces a Lorentz force on the cantilever free end, resulting in cantilever actuation. This same current flowing through a resistive heater induces a controllable temperature increase. We present cantilevers designed for large actuation forces for a given cantilever temperature increase. We analyze the designs of two new cantilevers, along with a legacy cantilever design. The cantilevers are designed to have a spring constant of about 1.5 N m(-1), a resonant frequency near 100 kHz, and self-heating capability with temperature controllable over the range 25-600 °C. Compared to previous reports on self-heating cantilevers, the Lorentz-thermal cantilevers generate up to seven times as much Lorentz force and two times as much oscillation amplitude. When used for thermal topography imaging, the Lorentz-thermal cantilevers can measure topography with a vertical resolution of 0.2 nm.