Deep-Learning-Based Nanomechanical Vibration for Rapid and Label-Free Assay of Epithelial Mesenchymal Transition.

Cancer is a profound danger to our life and health. The classification and related studies of epithelial and mesenchymal phenotypes of cancer cells are key scientific questions in cancer research. Here, we investigated cancer cell colonies from a mechanical perspective and developed an assay for classifying epithelial/mesenchymal cancer cell colonies using the biomechanical fingerprint in the form of "nanovibration" in combination with deep learning. The classification method requires only 1 s of vibration data and has a classification accuracy of nearly 92.5%. The method has also been validated for the screening of anticancer drugs. Compared with traditional methods, the method has the advantages of being nondestructive, label-free, and highly sensitive. Furthermore, we proposed a perspective that subcellular structure influences the amplitude and spectrum of nanovibrations and demonstrated it using experiments and numerical simulation. These findings allow internal changes in the cell colony to be manifested by nanovibrations. This work provides a perspective and an ancillary method for cancer cell phenotype diagnosis and promotes the study of biomechanical mechanisms of cancer progression.

Detection of organic dyes using Ag NPAs/SMP SERS substrate produced via sandpaper template-assisted lithography and liquid-liquid interface self-assembly.

Surface-enhanced Raman spectroscopy (SERS) is a highly sensitive and reliable fingerprinting technique. However, its analytical capability is closely related to the quality of a SERS substrate used for the analysis. In particular, conventional colloidal substrates possess disadvantages in terms of controllability, stability, and reproducibility, which limit their application. In order to address these issues, a simple, cost-effective, and efficient SERS substrate based on silver nanoparticle arrays (Ag NPAs) and sandpaper-molded polydimethylsiloxane (SMP) was proposed in this work. Successfully prepared via template lithography and liquid-liquid interface self-assembly (LLISA), the substrate can be applied to the specific detection of organic dyes in the environment. The substrate exhibited good SERS performance, and the limit of detection (LOD) of rhodamine 6G (R6G) was shown to be 10-7 M under the optimal conditions (1000 grit sandpaper) with a relative standard deviation (RSD) of 7.76%. Moreover, the SERS signal intensity was maintained at 60% of the initial intensity after the substrate was stored for 30 days. In addition, the Ag NPAs/SMP SERS substrate was also employed to detect crystal violet (CV) and methylene blue (MB) with the LODs of 10-6 M and 10-7 M, respectively. In summary, the Ag NPAs/SMP SERS substrate prepared in this study has great potential for the detection of organic dyes in ecological environments.

Dynamic response of the cell traction force to osmotic shock.

Osmotic pressure is vital to many physiological activities, such as cell proliferation, wound healing and disease treatment. However, how cells interact with the extracellular matrix (ECM) when subjected to osmotic shock remains unclear. Here, we visualize the mechanical interactions between cells and the ECM during osmotic shock by quantifying the dynamic evolution of the cell traction force. We show that both hypertonic and hypotonic shocks induce continuous and large changes in cell traction force. Moreover, the traction force varies with cell volume: the traction force increases as cells shrink and decreases as cells swell. However, the direction of the traction force is independent of cell volume changes and is always toward the center of the cell-substrate interface. Furthermore, we reveal a mechanical mechanism in which the change in cortical tension caused by osmotic shock leads to the variation in traction force, which suggests a simple method for measuring changes in cell cortical tension. These findings provide new insights into the mechanical force response of cells to the external environment and may provide a deeper understanding of how the ECM regulates cell structure and function. Traction force exerted by cells under hypertonic and hypotonic shocks. Scale bar, 200 Pa. Color bar, Pa. The black arrows represent the tangential traction forces.

Fabrication of Micro-Cantilever Sensor Based on Clay Minerals for Humidity Detection.

In this paper, novel humidity sensors based on montmorillonite, kaolinite, and composite films coated on micro-cantilevers were prepared to measure the relative humidity (RH) values by the deflection of a micro-cantilever (MC) at room temperature. The humidity-sensing properties, such as response and recovery, sensitivity, repeatability, humidity hysteresis, and long-term stability, were investigated in the range of working humidity (10-80% RH). The humidity response in the close humidity range of 10% RH to 80% RH revealed a linear increase in water absorption of montmorillonite, kaolinite, and montmorillonite/kaolinite mixed dispersant (1:1) as a function of RH with linear correlation factors between the humidity change and deflection estimated to be 0.994, 0.991, and 0.946, respectively. Montmorillonite's sensitivity was better than kaolinite's, with the mixed-clay mineral film's response falling somewhere in between. This research provides a feasible and effective approach to constructing high-performance MC humidity sensors that can be operated at room temperature based on clay minerals.

Nanomechanical assay for ultrasensitive and rapid detection of SARS-CoV-2 based on peptide nucleic acid.

The massive global spread of the COVID-19 pandemic makes the development of more effective and easily popularized assays critical. Here, we developed an ultrasensitive nanomechanical method based on microcantilever array and peptide nucleic acid (PNA) for the detection of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) RNA. The method has an extremely low detection limit of 0.1 fM (105 copies/mL) for N-gene specific sequence (20 bp). Interestingly, it was further found that the detection limit of N gene (pharyngeal swab sample) was even lower, reaching 50 copies/mL. The large size of the N gene dramatically enhances the sensitivity of the nanomechanical sensor by up to three orders of magnitude. The detection limit of this amplification-free assay method is an order of magnitude lower than RT-PCR (500 copies/mL) that requires amplification. The non-specific signal in the assay is eliminated by the in-situ comparison of the array, reducing the false-positive misdiagnosis rate. The method is amplification-free and label-free, allowing for accurate diagnosis within 1 h. The strong specificity and ultra-sensitivity allow single base mutations in viruses to be distinguished even at very low concentrations. Also, the method remains sensitive to fM magnitude lung cancer marker (miRNA-155). Therefore, this ultrasensitive, amplification-free and inexpensive assay is expected to be used for the early diagnosis of COVID-19 patients and to be extended as a broad detection tool. Electronic Supplementary Material: Supplementary material (experimental section, N gene sequences and all nucleic acid sequences used in the study, Figs. S1-S6, and Tables S1-S3) is available in the online version of this article at 10.1007/s12274-022-4333-3.

Relay-type sensing mode: A strategy to push the limit on nanomechanical sensor sensitivity based on the magneto lever.

Ultrasensitive molecular detection and quantization are crucial for many applications including clinical diagnostics, functional proteomics, and drug discovery; however, conventional biochemical sensors cannot satisfy the stringent requirements, and this has resulted in a long-standing dilemma regarding sensitivity improvement. To this end, we have developed an ultrasensitive relay-type nanomechanical sensor based on a magneto lever. By establishing the link between very weak molecular interaction and five orders of magnitude larger magnetic force, analytes at ultratrace level can produce a clearly observable mechanical response. Initially, proof-of-concept studies showed an improved detection limit up to five orders of magnitude when employing the magneto lever, as compared with direct detection using probe alone. In this study, we subsequently demonstrated that the relay-type sensing mode was universal in application ranging from micromolecule to macromolecule detection, which can be easily extended to detect enzymes, DNA, proteins, cells, viruses, bacteria, chemicals, etc. Importantly, we found that, sensitivity was no longer subject to probe affinity when the magneto lever was sufficiently high, theoretically, even reaching single-molecule resolution. Electronic Supplementary Material: Supplementary material (experimental section) is available in the online version of this article at 10.1007/s12274-022-5049-0.

Magneto-Nanomechanical Array Biosensor for Ultrasensitive Detection of Oncogenic Exosomes for Early Diagnosis of Cancers.

Exosomes are a class of nanoscale vesicles secreted by cells, which contain abundant information closely related to parental cells. The ultrasensitive detection of cancer-derived exosomes is highly significant for early non-invasive diagnosis of cancer. Here, an ultrasensitive nanomechanical sensor is reported, which uses a magnetic-driven microcantilever array to selectively detect oncogenic exosomes. A magnetic force, which can produce a far greater deflection of microcantilever than that produced by the intermolecular interaction force even with very low concentrations of target substances, is introduced. This method reduced the detection limit to less than 10 exosomes mL-1 . Direct detection of exosomes in the serum of patients with breast cancer and in healthy people showed a significant difference. This work improved the sensitivity by five orders of magnitude as compared to that of traditional nanomechanical sensing based on surface stress mode. This method can be applied parallelly for highly sensitive detection of other microorganisms (such as bacteria and viruses) by using different probe molecules, which can provide a supersensitive detection approach for cancer diagnosis, food safety, and SARS-CoV-2 infection.

Noise analysis in Stokes parameter reconstruction for division-of-focal-plane polarimeters.

The division-of-focal-plane (DoFP) polarimeter can quickly and effectively obtain the polarization information of light in real time, where Stokes parameter reconstruction is a critical issue. Many reconstruction methods have been proposed to address this; however, their performance tends to degrade in the presence of noise. Thus, it is significant to clarify the noise-induced error in Stokes parameter reconstruction. In this work, we investigate the link between the noise-introduced error and the reconstruction method and develop a simple and effective way to evaluate the noise robustness of reconstruction methods. Furthermore, a novel experimental scheme of noise measurement, to the best of our knowledge, is designed to verify the theory. Based on the criterion, our scheme guides the selection of reconstruction methods and further promotes the practical application of the DoFP technique.

Magnetic nanocomposite hydrogel with tunable stiffness for probing cellular responses to matrix stiffening.

As cells have the capacity to respond to their mechanical environment, cellular biological behaviors can be regulated by the stiffness of extracellular matrix. Moreover, biological processes are dynamic and accompanied by matrix stiffening. Herein, we developed a stiffening cell culture platform based on polyacrylamide-Fe3O4 magnetic nanocomposite hydrogel with tunable stiffness under the application of magnetic field. This platform provided a wide range of tunable stiffness (∼0.3-20 kPa) covering most of human tissue elasticity with a high biocompatibility. Overall, the increased magnetic interactions between magnetic nanoparticles reduced the pore size of the hydrogel and enhanced the hydrogel stiffness, thereby facilitating the adhesion and spreading of stem cells, which was attributed to the F-actin assembly and vinculin recruitment. Such stiffening cell culture platform provides dynamic mechanical environments for probing the cellular response to matrix stiffening, and benefits studies of dynamic biological processes. STATEMENT OF SIGNIFICANCE: Cellular biological behaviors can be regulated by the stiffness of extracellular matrix. Moreover, biological processes are dynamic and accompanied by matrix stiffening. Herein, we developed a stiffening cell culture platform based on polyacrylamide/Fe3O4 magnetic nanocomposite hydrogels with a wide tunable range of stiffness under the application of magnetic field, without adversely affecting cellular behaviors. Such matrix stiffening caused by enhanced magnetic interaction between magnetic nanoparticles under the application of the magnetic field could induce the morphological variations of stem cells cultured on the hydrogels. Overall, our stiffening cell culture platform can be used not only to probe the cellular response to matrix stiffening but also to benefit various biomedical studies.

Nanomechanical sensor for rapid and ultrasensitive detection of tumor markers in serum using nanobody.

Early cancer diagnosis requires ultrasensitive detection of tumor markers in blood. To this end, we develop a novel microcantilever immunosensor using nanobodies (Nbs) as receptors. As the smallest antibody (Ab) entity comprising an intact antigen-binding site, Nbs achieve dense receptor layers and short distances between antigen-binding regions and sensor surfaces, which significantly elevate the generation and transmission of surface stress. Owing to the inherent thiol group at the C-terminus, Nbs are covalently immobilized on microcantilever surfaces in directed orientation via one-step reaction, which further enhances the stress generation. For microcantilever-based nanomechanical sensor, these advantages dramatically increase the sensor sensitivity. Thus, Nb-functionalized microcantilevers can detect picomolar concentrations of tumor markers with three orders of magnitude higher sensitivity, when compared with conventional Ab-functionalized microcantilevers. This proof-of-concept study demonstrates an ultrasensitive, label-free, rapid, and low-cost method for tumor marker detection. Moreover, interestingly, we find Nb inactivation on sensor interfaces when using macromolecule blocking reagents. The adsorption-induced inactivation is presumably caused by the change of interfacial properties, due to binding site occlusion upon complex coimmobilization formations. Our findings are generalized to any coimmobilization methodology for Nbs and, thus, for the construction of high-performance immuno-surfaces. Electronic Supplementary Material: Supplementary material (experimental section, HER2 detection using anti-HER2-mAb-functionalized microcantilevers) is available in the online version of this article at 10.1007/s12274-021-3588-4.

Video microscopy-based accurate optical force measurement by exploring a frequency-changing sinusoidal stimulus.

Optical tweezers are constantly evolving micromanipulation tools that can provide piconewton force measurement accuracy and greatly promote the development of bioscience at the single-molecule scale. Consequently, there is an urgent need to characterize the force field generated by optical tweezers in an accurate, cost-effective, and rapid manner. Thus, in this study, we conducted a deep survey of optically trapped particle dynamics and found that merely quantifying the response amplitude and phase delay of particle displacement under a sine input stimulus can yield sufficiently accurate force measurements. In addition, Nyquist-Shannon sampling theorem suggests that the entire recovery of the accessible particle sinusoidal response is possible, provided that the sampling theorem is satisfied, thereby eliminating the requirement for high-bandwidth (typically greater than 10 kHz) detectors. Based on this principle, we designed optical trapping experiments by loading a sinusoidal signal into the optical tweezers system and recording the trapped particle responses with 45 frames per second (fps) charge-coupled device (CCD) and 163 fps complementary metal-oxide-semiconductor (CMOS) cameras for video microscopy imaging. The experimental results demonstrate that the use of low-bandwidth detectors is suitable for highly accurate force quantification, thereby greatly reducing the complexity of constructing optical tweezers. The trap stiffness increases significantly as the frequency increases, and the experimental results demonstrate that the trapped particles shifting along the optical axis boost the transversal optical force.

Regulating trapping energy for multi-object manipulation by random phase encoding.

As known to all, optical tweezers depend intensely on trapping laser power. Therefore, the ability to separately regulate trapping power for each optical trap under a multi-object manipulation task empowers researchers with more flexibility and possibilities. Here, we introduce a simple strategy using complementary random binary phase design to achieve trapping energy assignment. The trap energy ratio can be expediently regulated by effective pixel numbers of the phase mask. We demonstrate the effectiveness and functionality of this approach by calibrating trap stiffness and directly measuring trapping power of each optical trap. In addition, we show the capability of rotating micro-beads with controlled speed and direction by supplying vortex beams with different energy ratios at specified positions. Our results imply that regulating the trap energy ratio will be of great significance in various applications, such as optical sorting and microfluidic scenarios.

Theoretical analysis on performance of digital speckle pattern: uniqueness, accuracy, precision, and spatial resolution.

The performance of digital image correlation is closely associated with the quality of speckle pattern. In this paper, the performance of digital speckle pattern is analyzed theoretically concerning four critical factors: uniqueness, accuracy, precision, and spatial resolution. Pattern uniqueness could be characterized by secondary autocorrelation peak height; based on a theoretical analysis on autocorrelation function of digital speckle pattern, analytical formulas are derived to estimate the secondary autocorrelation peak height. Measurement accuracy and precision are descriptions of systematic error and random error respectively; by deriving analytical expression for power spectrum of digital speckle pattern, theoretical models are built for analyzing both systematic errors and random errors. Spatial resolution characterizes the ability of a given technique to distinguish close features; empirical formulas are presented to describe the dependence of spatial resolution upon subset size and shape function order; besides, a rudimentary model is proposed, which provides conservative estimates for spatial resolution. Considering all these four factors, we make recommendations for selection of generation parameters of digital speckle pattern, which can eventually improve the measurement performance of digital image correlation technique.

Label-free biosensing using a microring resonator integrated with poly-(dimethylsiloxane) microfluidic channels.

Microring resonators have shown promising potential for highly sensitive, label-free, real-time detection of biomolecules. Accurate quantitative detection of target molecules through use of photonic integrated circuits has been demonstrated for environmental monitoring and medical diagnostics. Here, we described the design, fabrication, and characterization of a highly sensitive, label-free microring optical resonator integrated with poly-(dimethylsiloxane) microfluidic channels, which consumes only 30 µl of sample solution. The resonance wavelength shifts resulting from the change in the effective refraction index can be measured in situ, and thus the binding events on the resonator surface, including antibody immobilization, blocking of the resonator surface, and the specific binding of antibody and antigen, can be recorded throughout the entire experimental process in real time. We measured the binding events for the detection of human immunoglobulin G. The system had a detection limit of 0.5 µg/ml, a value substantially (14 times) lower than that of a previously reported microring resonator. To verify the usefulness and adaptability of this technique, human epidermal growth factor receptor 2 was used for the detection. The microring optical resonator was able to monitor reactions between biological molecules in real time and thus can be used in quantitative detection and biological sensing with little sample consumption.

Aptamer-based microcantilever-array biosensor for profenofos detection.

Profenofos, a highly poisonous organophosphorus pesticide, has been widely used in agricultural production. These pesticide residues have seriously influenced food security and threatened human health, and new methods with high sensitivity are greatly needed to detect profenofos. Here, we developed an aptamer-based microcantilever-array sensor operated in stress mode to detect profenofos, with advantages of being a label-free, highly sensitive, one-step immobilization method capable of quantitative and real-time detection. The microcantilevers were functionalized with a profenofos-specific aptamer (SS2-55), and then the specific binding of profenofos to aptamer induced a deflection of the microcantilever, which was monitored using an optical method in a real-time manner. The microcantilever deflection showed a positive relationship with profenofos concentration, and the detection limit was low to 1.3 ng mL-1 (3.5 nM) for profenofos, which was much lower than other aptamer-based detection methods. The selectivity of the sensor was verified with another organophosphorus pesticide. Additionally, we successfully detected profenofos dissolved in vegetable-soak solution. Our results showed that this aptamer-based microcantilever-array sensor is a convenient and label-free method for detecting profenofos in small amounts and has great potential for food-security applications.

Nanomechanical sensors for direct and rapid characterization of sperm motility based on nanoscale vibrations.

Infertility, whether of male or female origin, is a critical challenge facing the low birth rate and aging population throughout the world, and semen analysis is a cornerstone of the diagnostic evaluation of the male contribution to infertility. This means that tools which can characterize sperm properties in an effective manner are very much needed. The conventional approaches are essentially image-based, which have a limited value for analyzing sperm properties. Here, we show that an assay using nanomechanical sensors can detect sperm motility based on nanomotion. We use microcantilever sensors to directly characterize the mechanical response of the sperm based on the fluctuations of microcantilevers. We applied this methodology to sperms exposed to different chemical or physical agents. Real-time nanomechanical fluctuations showed that living sperms produced smaller fluctuations after treatment with inhibitory chemicals, and larger fluctuations after treatment with stimulatory chemicals. Our preliminary experiments suggest that the frequency of fluctuation is associated with sperm motility. This technique offers a brand-new perspective in the characterization of the sperm. By combining conventional measurements, reproductive medicine doctors and researchers should now be able to achieve unprecedented depth in the sperm properties.

Microcantilever array instrument based on optical fiber and performance analysis.

We developed a microcantilever array biosensor instrument based on optical readout from a microcantilever array in fluid environment. The microcantilever signals were read out sequentially by laser beams emitted from eight optical fibers. The optical fibers were coupled to lasers, while the other ends of the fibers were embedded in eight V-grooves with 250 µm pitch microfabricated from a Si wafer. Aspherical lens was used to keep the distance between lasers. A programmable logic controller was used to make the system work stably. To make sure that the output of lasers was stable, a temperature controller was set up for each laser. When the deflection signal was collected, lasers used here were set to be on for at least 400 ms in each scanning cycle to get high signal-to-noise ratio deflection curves. A test was performed by changing the temperature of the liquid cell holding a microcantilever array to verify the consistent response of the instrument to the cantilever deflections. The stability and conformance of the instrument were demonstrated by quantitative detection of mercury ions in aqueous solution and comparison detection of clenbuterol by setting test and reference cantilevers. This microcantilever array detection instrument can be applied to highly sensitive detection of chemical and biological molecules in fluid environment.

Measurement of Airy-vortex beam topological charges based on a pixelated micropolarizer array.

In this paper, we numerically studied the intensity patterns and screw phases of an embedded optical vortex in an Airy beam generated by a 3/2 phase pattern imposed on a spatial light modulator. It is found that the optical vortex and the Airy beam's main lobe approach each other during propagation, which means the energy of the Airy beam's intensity peaks can be taken advantage of by the imposed vortices. Based on a pixelated micropolarizer array in the interference path, we succeeded in measuring the integer topological charges up to -10 according to the phase jump. In addition, fractional topological charges were also obtained in the experiment. Both of the experimental results are acquired in a high-precision and robust way. This work will promote potential application of Airy-vortex beams in fields such as optical manipulation, laser processing, and photon entanglement.

Quantification of cell viability and rapid screening anti-cancer drug utilizing nanomechanical fluctuation.

Cancer is a serious threat to human health. Although numerous anti-cancer drugs are available clinically, many have shown toxic side effects due to poor tumor-selectivity, and reduced effectiveness due to cancers rapid development of resistance to treatment. The development of new highly efficient and practical methods to quantify cell viability and its change under drug treatment is thus of significant importance in both understanding of anti-cancer mechanism and anti-cancer drug screening. Here, we present an approach of utilizing a nanomechanical fluctuation based highly sensitive microcantilever sensor, which is capable of characterizing the viability of cells and quantitatively screening (within tens of minutes) their responses to a drug with the obvious advantages of a rapid, label-free, quantitative, noninvasive, real-time and in-situ assay. The microcantilever sensor operated in fluctuation mode was used in evaluating the paclitaxel effectiveness on breast cancer cell line MCF-7. This study demonstrated that the nanomechanical fluctuations of the microcantilever sensor are sensitive enough to detect the dynamic variation in cellular force which is provided by the cytoskeleton, using cell metabolism as its energy source, and the dynamic instability of microtubules plays an important role in the generation of the force. We propose that cell viability consists of two parts: biological viability and mechanical viability. Our experimental results suggest that paclitaxel has little effect on biological viability, but has a significant effect on mechanical viability. This new method provides a new concept and strategy for the evaluation of cell viability and the screening of anti-cancer drugs.

Highly sensitive nanomechanical immunosensor using half antibody fragments.

The improvement of sensitivity is of great significance to the application of biochemical sensor. In this study, we propose a micocantilever-based immunosensor in surface stress mode using half antibody fragments as receptor molecules. The thiol-containing half antibody fragment was obtained with a low loss of antibody biological activity and then was covalently and orientedly immobilized on the gold surface of microcantilevers via two native thiol groups. Such a one-step reaction and immobilization of receptor molecule simplify the preparation process of micocantilever immunosensor. Using shortened and highly oriented half antibody fragments as receptor molecules, the generation of surface stress and the transmission of stress from the interaction region of molecules to the surface of the microcantilever have been elevated significantly. The limit of detection (LOD) of the presented sensor has been significantly lowered to 1 pg/mL, or 1.1 pM in equivalence, which is a 500-fold improvement when compared with intact full antibody coated conventional micocantilever sensors. The results indicate that the half antibody fragment is well suited for the functionalization of the microcantilever surface and is generally applicable to all microcantilever immunosensor development, and this principle will help to design a functional film of devices with significantly lower LOD.