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1.
Sensors (Basel) ; 24(19)2024 Oct 06.
Artículo en Inglés | MEDLINE | ID: mdl-39409498

RESUMEN

Developing powerful immunoassays for sensitive and real-time detection of targets has always been a challenging task. Due to their advantages of direct readout, controllable sensing, and low background interference, photothermal immunoassays have become a type of new technology that can be used for various applications such as disease diagnosis, environmental monitoring, and food safety. By modification with antibodies, photothermal materials can induce temperature changes by converting light energy into heat, thereby reporting specific target recognition events. This article reviews the design and application of photothermal immunoassays based on different photothermal materials, including noble metal nanomaterials, carbon-based nanomaterials, two-dimensional nanomaterials, metal oxide and sulfide nanomaterials, Prussian blue nanoparticles, small organic molecules, polymers, etc. It pays special attention to the role of photothermal materials and the working principle of various immunoassays. Additionally, the challenges and prospects for future development of photothermal immunoassays are briefly discussed.


Asunto(s)
Técnicas Biosensibles , Inmunoensayo/métodos , Humanos , Técnicas Biosensibles/métodos , Nanoestructuras/química , Temperatura , Ferrocianuros/química
2.
Molecules ; 29(12)2024 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-38930860

RESUMEN

Enzyme-linked electrochemical immunosensors have attracted considerable attention for the sensitive and selective detection of various targets in clinical diagnosis, food quality control, and environmental analysis. In order to improve the performances of conventional immunoassays, significant efforts have been made to couple enzyme-linked or nanozyme-based catalysis and redox cycling for signal amplification. The current review summarizes the recent advances in the development of enzyme- or nanozyme-based electrochemical immunosensors with redox cycling for signal amplification. The special features of redox cycling reactions and their synergistic functions in signal amplification are discussed. Additionally, the current challenges and future directions of enzyme- or nanozyme-based electrochemical immunosensors with redox cycling are addressed.


Asunto(s)
Técnicas Biosensibles , Técnicas Electroquímicas , Oxidación-Reducción , Técnicas Biosensibles/métodos , Técnicas Electroquímicas/métodos , Inmunoensayo/métodos , Catálisis , Humanos , Enzimas/metabolismo , Enzimas/química
3.
Anal Chem ; 95(8): 4227-4234, 2023 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-36795965

RESUMEN

Fluorescence-assisted digital counting analysis allowed sensitive quantification of targets by measuring individual fluorescent labels. However, traditional fluorescent labels suffered from low brightness, small size, and sophisticated preparation procedures. Herein, engineering fluorescent dye-stained cancer cells with magnetic nanoparticles were proposed to construct single-cell probes for fluorescence-assisted digital counting analysis by quantifying the target-dependent binding or cleaving events. Various engineering strategies of cancer cells including biological recognition and chemical modification were developed for rationally designing single-cell probes. Introduction of suitable recognition elements into single-cell probes allowed digital quantification of each target-dependent event via counting the colored single-cell probes in the representative image taken using a confocal microscope. The reliability of the proposed digital counting strategy was corroborated by traditional optical microscopy- and flow cytometry-dependent counting technologies. The advantages of single-cell probes, including high brightness, big size, ease of preparation, and magnetic separation, contributed to the sensitive and selective analysis of targets of interest. As proof-to-concept assays, indirect analysis of exonuclease III (Exo III) activity, as well as direct quantitation of cancer cells, were investigated, and the potential in biological sample analysis was also assessed. This sensing strategy will open a new avenue for the development of biosensors.


Asunto(s)
Técnicas Biosensibles , Nanopartículas , Neoplasias , Reproducibilidad de los Resultados , Espectrometría de Fluorescencia , Técnicas Biosensibles/métodos , Colorantes Fluorescentes/metabolismo , Sondas de ADN
4.
Opt Lett ; 48(7): 1846-1849, 2023 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-37221781

RESUMEN

In this Letter, we report a bridge-connected three-electrode germanium-on-silicon (Ge-on-Si) avalanche photodiode (APD) array compatible with the complementary metal-oxide semiconductor (CMOS) process. In addition to the two electrodes on the Si substrate, a third electrode is designed for Ge. A single three-electrode APD was tested and analyzed. By applying a positive voltage on the Ge electrode, the dark current of the device can be reduced, and yet the response of the device can be increased. Under a dark current of 100 nA, as the voltage on Ge increases from 0 V to 15 V, the light responsivity is increased from 0.6 A/W to 1.17 A/W. We report, for the first time to the best of our knowledge, the near-infrared imaging properties of an array of three-electrode Ge-on-Si APDs. Experiments show that the device can be used for LiDAR imaging and low-light detection.

5.
Molecules ; 28(12)2023 Jun 14.
Artículo en Inglés | MEDLINE | ID: mdl-37375313

RESUMEN

Poly(ADP-ribose) polymerase-1 (PARP1) is a potential biomarker and therapeutic target for cancers that can catalyze the poly-ADP-ribosylation of nicotinamide adenine dinucleotide (NAD+) onto the acceptor proteins to form long poly(ADP-ribose) (PAR) polymers. Through integration with aggregation-induced emission (AIE), a background-quenched strategy for the detection of PARP1 activity was designed. In the absence of PARP1, the background signal caused by the electrostatic interactions between quencher-labeled PARP1-specitic DNA and tetraphenylethene-substituted pyridinium salt (TPE-Py, a positively charged AIE fluorogen) was low due to the fluorescence resonance energy transfer effect. After poly-ADP-ribosylation, the TPE-Py fluorogens were recruited by the negatively charged PAR polymers to form larger aggregates through electrostatic interactions, thus enhancing the emission. The detection limit of this method for PARP1 detection was found to be 0.006 U with a linear range of 0.01~2 U. The strategy was used to evaluate the inhibition efficiency of inhibitors and the activity of PARP1 in breast cancer cells with satisfactory results, thus showing great potential for clinical diagnostic and therapeutic monitoring.


Asunto(s)
NAD , Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/metabolismo , Electricidad Estática , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , NAD/metabolismo
6.
BMC Nurs ; 22(1): 128, 2023 Apr 18.
Artículo en Inglés | MEDLINE | ID: mdl-37072844

RESUMEN

BACKGROUND: Providing self-management support to kidney transplant recipients is essential. However, a scale to identify the self-management support they have received is lacking. The purpose of this study is to develop a Self-management Support Scale for Kidney Transplant Recipients (SMSSKTR) and test its psychometric properties. METHODS: This is an instrument development and validation study, which has a three-stage cross-sectional design. In Stage 1, a preliminary item pool was formed using a literature review, semi-structured interviews, and the Delphi method. In Stage 2, six experts were invited to assess content validity. A convenience sample of 313 participants was used to explore the factor structure by using exploratory factor analysis. The test-retest reliability was assessed using the intra-class correlation coefficient (ICC). In Stage 3, two hundred and sixty-five participants were recruited to validate the factor structure by using confirmatory factor analysis. Convergent validity was examined using Spearman's correlation coefficient. Cronbach's alpha coefficient and corrected item-total correlation coefficient were used to test the reliability of the entire scale and its dimensions. The study was reported according to the STARD and GRRAS checklists. RESULTS: An initial 40-item scale was developed in Stage 1. In Stage 2, three factors with 22 items emerged from the exploratory factor analysis: instrumental support, psychosocial support, and relational support. The content validity index of the scale was 0.97. The intra-class correlation coefficient for the entire scale and the subscales were 0.915, 0.771, 0.896, and 0.832, respectively. In Stage 3, the confirmatory factor analysis indicated that the three-factor model had a good fit. The score of the scale was positively associated with that of the Self-Management Scale of Renal Transplant Recipients (r = 0.532). Cronbach's alpha was 0.959 for the entire scale and 0.956-0.958 for the three subscales. The corrected item-total correlation coefficient ranged from 0.62 to 0.82. CONCLUSION: The 22-item SMSSKTR has sufficient psychometric properties to assess the self-management support they have received, which has not been measured before.

7.
Angew Chem Int Ed Engl ; 62(24): e202303812, 2023 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-37069482

RESUMEN

The induction of diverse chirality regulation in nature by multiple binding sites of biomolecules is ubiquitous and plays an essential role in determining the biofunction of biosystems. However, mimicking this biological phenomenon and understanding at a molecular level its mechanism with the multiple binding sites by establishing an artificial system still remains a challenge. Herein, abundant chirality inversion is achieved by precisely and multiply manipulating the co-assembled binding sites of phenylalanine derivatives (D/LPPF) with different naphthalene derivatives (NA, NC, NP, NF). The amide and hydroxy group of naphthalene derivatives prefer to bind with the carboxy group of LPPF, while carboxylic groups and fluoride atoms tend to bind with the amide moiety of LPPF. All these diverse interaction modes can precisely trigger helicity inversion of LPPF nanofibers. In addition, synergistically manipulating the carboxy and amide binding sites from a single LPPF molecule to simultaneously interact with different naphthalene derivatives leads to chirality regulation. Typically, varying the solvent may switch the interaction modes and the switched new interaction modes can be employed to further regulate the chirality of the LPPF nanofibers. This study may provide a novel approach to explore chirality diversity in artificial systems by regulating the intermolecular binding sites.

8.
Opt Express ; 30(12): 22221-22232, 2022 Jun 06.
Artículo en Inglés | MEDLINE | ID: mdl-36224925

RESUMEN

Traditional free-space laser communication systems use beacon and signal lights for target detection and alignment. However, these approaches are inaccurate owing to signal dispersion errors. To overcome this difficulty, we propose a new method using transient radio frequency (RF) signals to achieve highly accurate target detection and alignment. To validate the feasibility of our proposed method, we built an experimental multi-target space-laser communication system based on a rotating double prism and applied it to achieve multi-target space-laser communication. The results demonstrate the efficiency of the proposed method to capture multi-target positions in the field of view using wireless RF signals and a rotating double prism. In addition, we show that the system is capable of rapid scanning and accurate pointing as well as establishing a one-way stable communication with multiple targets. When the target is 36 cm away, the pointing accuracy of the system motor is less than 0.8°, the pointing time is 1.2 s, and the average pointing lateral error is 0.666 mm.

9.
Opt Express ; 30(4): 5008-5018, 2022 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-35209472

RESUMEN

Silicon based optoelectronic integrated optical phased array is attractive owing to large-dense integration, large scanning range and CMOS compatibility. In this paper, we design and fabricate a SiN-on-SOI two-dimensional optical phased array chip. We demonstrate a two-dimensional scanning range of 96°×14.4° and 690 mW peak power of the main lobe. Additionally, we set up the time of flight (ToF) and frequency-modulated continuous-wave (FMCW) ranging systems by using this optical phased array chip, and achieve the objects detection at the range of 20 m in the ToF system and 109 m in the FMCW system, respectively.

10.
Sensors (Basel) ; 22(21)2022 Oct 23.
Artículo en Inglés | MEDLINE | ID: mdl-36365813

RESUMEN

It has become routine to directly process point clouds using a combination of shared multilayer perceptrons and aggregate functions. However, this practice has difficulty capturing the local information of point clouds, leading to information loss. Nevertheless, several recent works have proposed models that establish point-to-point relationships based on this procedure. However, to address the information loss, in this study we use self-supervised methods to enhance the network's understanding of point clouds. Our proposed multigrid autoencoder (MA) constrains the encoder part of the classification network so that it gains an understanding of the point cloud as it reconstructs it. With the help of self-supervised learning, we find the original network improves performance. We validate our model on PointNet++, and the experimental results show that our method improves overall classification accuracy by 2.0% and 4.7% with ModelNet40 and ScanObjectNN datasets, respectively.

11.
Int J Nurs Pract ; 28(5): e13085, 2022 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-35903948

RESUMEN

AIM: To investigate the current condition and degree of fear of disease progression and associated factors in patients with mild or common type COVID-19. BACKGROUND: At the end of 2019, COVID-19 spread from Wuhan in Hubei Province throughout China. Confirmed cases and deaths have since been reported in many countries around the world. However, fear of progression in these patients has been poorly explored. METHODS: During February 2020, we recruited 114 patients with mild or common type COVID-19 admitted to a Fangcang shelter hospital. We assessed patients' degree of fear using the simplified Fear of Progression Questionnaire (Chinese version). Multiple regression analysis was applied to explore potential factors. RESULTS: The fear of disease progression scores of patients with mild or common COVID-19 was at the low-to-moderate level. Current unemployment, disease duration of 28 days or more and not having a spouse diagnosed with COVID-19 were factors potentially associated with fear of progression. CONCLUSION: With a high prevalence of fear of disease progression in patients with COVID-19, the risk of psychological effects from the pandemic is significant and fear of progression is one of the manifestations. The need for psychological support services for patients should be included in all pandemic and disaster planning.


Asunto(s)
COVID-19 , COVID-19/epidemiología , China/epidemiología , Miedo , Hospitales Especializados , Humanos , Unidades Móviles de Salud , Trastornos Fóbicos , SARS-CoV-2
12.
Opt Express ; 29(13): 20995-21010, 2021 Jun 21.
Artículo en Inglés | MEDLINE | ID: mdl-34266175

RESUMEN

Two novel waveguide gratings for optical phased array transmitters are investigated. By offsetting the grating structures along the waveguide on the upper and lower surfaces of the silicon nitride (Si3N4) waveguide, the dual-level chain and dual-level fishbone structures can achieve 95% of unidirectional radiation with a single Si3N4 layer by design. With apodized perturbation along the gratings, both structures can achieve uniform radiation without compromising the unidirectional radiation performance. In experiment, both demonstrate ∼ 80-90% unidirectionality. With further analysis, it is found that the dual-level fishbone structure is more feasible and robust to process variations in uniform radiation.

13.
Opt Express ; 29(11): 16346-16361, 2021 May 24.
Artículo en Inglés | MEDLINE | ID: mdl-34154200

RESUMEN

To address the problem of traditional surface illuminated detectors being of low responsivity, this work proposes a large-size interdigitated "finger-type" germanium-on-silicon (Ge-on-Si) photodetector (PD) based on the surface illumination approach. For 1550 nm light with a surface incident power of -20 dBm at room temperature, the best responsivity of the PD achieved is ∼0.64 A/W at 0.5 V. At the same time, the optimal bandwidth reaches 1.537 MHz with 3.5 V applied voltage. In order to suppress the dark current induced noise, a Ge-on-Si avalanche photodiode (APD) with the interdigitated structure is designed. The avalanche voltage is designed ∼13.3 V at room temperature, and the dark current density in linear region is at mA/cm2 order. We believe this type of device can be applied in weak light detection condition.

14.
Opt Express ; 29(19): 29755-29765, 2021 Sep 13.
Artículo en Inglés | MEDLINE | ID: mdl-34614714

RESUMEN

The optical power handling of an OPA scanning beam determines its targeted detection distance. So far, a limited number of investigations have been conducted on the restriction of the beam power. To the best of our knowledge, we for the first time in this paper explore the ability of the silicon photonics based OPA circuit for the high power application. A 64-channel SiN-Si based one-dimensional (1D) OPA chip has been designed to handle high beam power to achieve large scanning range. The chip was fabricated on the standard silicon photonics platform. The main lobe power of our chip can reach 720 mW and its peak side-lobe level (PSLL) is -10.33 dB. We obtain a wide scanning range of 110° in the horizontal direction at 1550 nm wavelength, with a compressed longitudinal divergence angle of each scanning beam of 0.02°.

15.
Adv Skin Wound Care ; 34(10): 532-537, 2021 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-34546204

RESUMEN

OBJECTIVE: To explore the experience of incontinence-associated dermatitis (IAD) as perceived by nurses, obstacles in the nursing process, and need for IAD training and management. METHODS: This single-setting descriptive qualitative study was conducted from June 5, 2018, to June 22, 2018. Ten nurses working in the respiratory ICU of a local hospital participated in semistructured interviews. The content analysis method was used to analyze, summarize, and refine the interview data. RESULTS: The experience of ICU nurses with IAD can be divided into four types: nursing based on experiential knowledge, seeking self-improvement, disunity of cleaning methods and wiping skills, and postponement of nursing care because of priority allocation. Obstacles in the nursing process include a lack of relevant nursing knowledge and awareness, as well as the medical supplies needed. The goals of training and management include establishing IAD preventive nursing procedures, providing IAD care products, enhancing the practicality of training content and diversifying training methods, and establishing an information system to assist nurses caring for patients with IAD. CONCLUSIONS: The knowledge and behavior of ICU nurses regarding IAD need to be improved. Training and management are imperative. Facilities and nurse managers should actively seek solutions to stated obstacles, formulate training methods suitable for clinical needs, and promote the standardization of nursing for IAD.


Asunto(s)
Dermatitis por Contacto/etiología , Enseñanza/estadística & datos numéricos , Adulto , China , Educación Continua en Enfermería/métodos , Incontinencia Fecal/complicaciones , Incontinencia Fecal/fisiopatología , Femenino , Humanos , Unidades de Cuidados Intensivos/organización & administración , Unidades de Cuidados Intensivos/estadística & datos numéricos , Masculino , Persona de Mediana Edad , Investigación Cualitativa , Cuidados de la Piel/métodos , Cuidados de la Piel/normas , Cuidados de la Piel/estadística & datos numéricos , Enseñanza/normas , Incontinencia Urinaria/complicaciones , Incontinencia Urinaria/fisiopatología
16.
Exp Mol Pathol ; 113: 104373, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-31917285

RESUMEN

Asthma is a chronic inflammatory airway disease. Icariside II has been reported to exert anti-inflammatory effect in multiple human diseases. The present study aimed to investigate the effects and mechanisms of Icariside II on airway inflammation and remodeling in asthma. We established an asthma mouse model with ovalbumin (OVA) immunization. Histological analysis using H&E, PAS and Masson staining showed that administration of Icariside II attenuated OVA-induced airway inflammation and remodeling. Icariside II reduced the numbers of total white blood cells and eosinophils in bronchoalveolar lavage fluid (BALF). The levels of interleukin (IL)-4, IL-5, IL-13 and transforming growth factor (TGF)-ß1 in peripheral blood and the expression of α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), eotaxin-1, CC-chemokine receptor-3 (CCR-3), Toll-like receptor (TLR)-2 and TLR-4 were significantly down-regulated in lung tissues of OVA-induced mouse model. These results suggested that Icariside II inhibited eosinophil activation and thus decreased eosinophils-induced airway inflammation and remodeling in asthma. Moreover, Icariside II suppressed TGF-ß1-induced cell proliferation, migration, and CTGF expression in airway smooth muscle cells (ASMCs). In both OVA-induced mouse model of asthma and TGF-ß1-induced ASMCs, Icariside II decreased IκBα degradation, nuclear translocation of NF-κB p65 and STAT3 phophorylation, indicating an inactivation of NF-κB and STAT3 in the presence of Icariside II. Therefore, we demonstrate that Icariside II attenuates eosinophils-induced airway inflammation and remodeling in asthmatic mice and inhibits TGF-ß1-induced cell proliferation and migration in ASMCs via suppressing NF-κB and STAT3 signalings.


Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias) , Asma/tratamiento farmacológico , Eosinófilos/patología , Flavonoides/uso terapéutico , Inflamación/patología , Pulmón/patología , FN-kappa B/metabolismo , Factor de Transcripción STAT3/metabolismo , Remodelación de las Vías Aéreas (Respiratorias)/efectos de los fármacos , Animales , Asma/metabolismo , Asma/patología , Asma/fisiopatología , Líquido del Lavado Bronquioalveolar , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Modelos Animales de Enfermedad , Eosinófilos/efectos de los fármacos , Flavonoides/farmacología , Masculino , Ratones Endogámicos BALB C , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta1/farmacología
17.
Anal Chem ; 91(5): 3336-3341, 2019 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-30724069

RESUMEN

Many mitochondria-related diseases are associated with the mutation of mitochondrial DNA (mtDNA). Therefore, visualizing its dynamics in live cells is essential for the understanding of the function of mtDNA transcription and translation. By employing carbazole as the framework and designing a module for DNA minor-groove binding, here we have developed a novel fluorescent probe with a large Stokes shift (λab = 480 nm and λem = 620 nm), CNQ, for mtDNA detection and visualization. It is almost nonfluorescent in PBS buffer and exhibits 182-fold enhancement in fluorescence within 20 s after the application of mtDNA in the solution, with a detection limit of 55.1 µg/L. Using dual-color Hessian-structured illumination microscopy, we have demonstrated that CNQ-labeled mtDNA structures are distinct from those labeled by TFAM-EGFP. Finally, we have used two-photon confocal scanning microscopy (λex = 850 nm) to monitor the nondestructive doxorubicin-induced mtDNA damage in live cells.


Asunto(s)
Carbazoles/química , ADN Mitocondrial/metabolismo , Colorantes Fluorescentes/química , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , ADN Mitocondrial/química , Humanos , Células MCF-7 , Mitocondrias/metabolismo
18.
Opt Express ; 27(9): 12883-12898, 2019 Apr 29.
Artículo en Inglés | MEDLINE | ID: mdl-31052822

RESUMEN

In this work, thermo-optic tunable 4 × 4 cascaded multimode interference based integrated optical waveguide switching matrices are designed and fabricated using photopolymer lightwave circuits. The materials of the waveguide core and cladding are fluorinated epoxy-terminated copolycarbonate and polymethylmethacrylate, respectively. The driving power that controls matrices for binary encoding of different optical switching states are simulated and analyzed. The measured insertion loss of the actual chip is < 7.1 dB and the maximum crosstalk in adjacent channels is <-30 dB. The switching time is approximately 220 µs and the extinction ratio is obtained as 21.5 dB. This flexible encoding technique can be applied for achieving optical code-division multiple-access network coders.

19.
Opt Express ; 26(15): 19154-19170, 2018 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-30114176

RESUMEN

We propose a pattern-search-like algorithm to design an aperiodic optical phased array for extensive applications in light detection and ranging and free-space communication. The designed phased array with 128 isotropic elements achieves a scan range, peak side-lobe level, minimum beam width, and mean pitch of ± 82°, -14.34 dB, 0.062°, and 9.75 µm, respectively. To our knowledge, it has the widest steering range, narrowest divergence, and largest mean pitch for the same waveguide number. The minimum pitch can be greater than 2.67λ to avoid cross-coupling. The calculated relationship between the machine error and side-lobe level indicates that the designed structure has a higher error tolerance than its uniformly spaced counterpart.

20.
Sensors (Basel) ; 18(2)2018 Jan 24.
Artículo en Inglés | MEDLINE | ID: mdl-29364847

RESUMEN

Multi-frequency scanning near-field optical microscopy, based on a quartz tuning fork-probe (QTF-p) sensor using the first two orders of in-plane bending symmetrical vibration modes, has recently been developed. This method can simultaneously achieve positional feedback (based on the 1st in-plane mode called the low mode) and detect near-field optically induced forces (based on the 2nd in-plane mode called the high mode). Particularly, the high mode sensing performance of the QTF-p is an important issue for characterizing the tip-sample interactions and achieving higher resolution microscopic imaging but the related researches are insufficient. Here, we investigate the vibration performance of QTF-p at high mode based on the experiment and finite element method. The frequency spectrum characteristics are obtained by our homemade laser Doppler vibrometer system. The effects of the properties of the connecting glue layer and the probe features on the dynamic response of the QTF-p sensor at the high mode are investigated for optimization design. Finally, compared with the low mode, an obvious improvement of quality factor, of almost 50%, is obtained at the high mode. Meanwhile, the QTF-p sensor has a high force sensing sensitivity and a large sensing range at the high mode, indicating a broad application prospect for force sensing.

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