Electrostatic Force Microscopy (EFM), an extension of Atomic Force Microscopy (AFM), characterizes electrical properties at the nanoscale by detecting long range electrostatic force gradients. It enables simultaneous acquisition of topography and electrostatic information from biological samples. Dynamic electrical changes are fundamental across all life processes, from biomolecular charge transport to cellular electrophysiology. EFM's high resolution and nondestructive nature make it essential for revealing the physical mechanisms at biological interfaces. This review systematically elaborates the evolution of EFM principles, including optimized working modes tailored for biology, and analyzes its hierarchical applications, from biomolecules and subcellular structures to cells and pathological diagnosis. Cross scale dielectric correlations and current technical challenges are discussed, alongside advanced solutions such as heterodyne high harmonic detection and multimodal integration, offering a systematic perspective for EFM's translational development in biomedicine.

KPFM nanoscale mapping of surface potential and cellular charges, revealing bioelectrical properties in cells and biomolecules, bridging nanoscale electrical properties, from membrane potentials to protein interactions, with biological function.

Atomic force microscopy (AFM) has transcended its role as a mere provider of high-resolution imaging in medical research, catalysing a paradigm shift from 'morphological observation' to 'quantitative mechanics' and 'single molecule manipulation'. AFM enables us to directly decipher the 'mechanical language' of living systems, acquiring information on sample topography, mechanical properties, and molecular interactions under near physiological conditions with nanoscale resolution. This review systematically elaborates on how AFM, by providing quantitative, functional, and dynamic nanoscale data, is reshaping the understanding of disease mechanisms. It is also fostering novel precision medicine strategies guided by 'mechanobiology' in areas such as cardiovascular diseases, cancer, and pathogen recognition. AFM not only expands the dimensions of medical research but also provides unprecedented tools and perspectives for disease diagnosis, drug development, and cellular intervention.

Atomic Force Microscopy (AFM) based single molecule recognition has emerged as a transformative paradigm in nanoscale biology, enabling direct investigation of biological interactions under physiological conditions. This technology bridges critical gaps between structural characterization and functional analysis by providing unparalleled capabilities for quantifying binding forces, mapping molecular distributions, and resolving kinetic parameters at the single molecule level. The core methodology relies on precision probe functionalization strategies, such as oriented immobilization and polyethylene glycol (PEG) linkers, to ensure specific recognition while minimizing nonspecific interactions. Advanced techniques, including single molecule force spectroscopy (SMFS) and topography and recognition imaging (TREC), allow simultaneous acquisition of morphological and binding data with nanometer resolution, revealing insights into molecular organization and dynamics. Applications span diverse domains, from fundamental studies of antigen-antibody interactions and DNA-protein binding to clinical investigations of membrane receptor distributions in cancer cells and pathological diagnosis based on mechanical properties. Despite challenges in throughput, signal resolution, and standardization, integration with super-resolution microscopy and spectroscopic techniques demonstrates significant potential. Future developments emphasize multimodal correlation, artificial intelligence-assisted data analysis, and clinical translation toward nano biopsy, positioning AFM as a highly valuable and complementary platform for advancing molecular biophysics, drug discovery, and personalized medicine, notably due to its unique capability to combine nanoscale imaging with single-molecule force measurement on the same sample under physiological conditions.

Nucleolin, a protein overexpressed on the surface of cancer cells, has emerged as a promising therapeutic target due to its high affinity interactions with aptamers. This study localized nucleolin on lung cancer and normal cells at single-molecule resolution using the single molecule recognition imaging mode of Atomic Force Microscopy (AFM) with three aptamers: 9FU-AS1411, AS1411, and CRO. The results revealed abundant nucleolin expression on lung cancer cells, while minimal levels were detected on normal cells. The binding affinities and interaction dynamics of these aptamers were systematically evaluated. Flow cytometry and AFM-based force spectroscopy demonstrated that 9FU-AS1411 exhibited the strongest unbinding forces (piconewton level) and higher dissociation activation energy compared to AS1411, indicating enhanced complex stability. In contrast, CRO showed negligible binding, confirming its lack of specificity. Further analysis via Kelvin Probe Force Microscopy (KPFM) revealed distinct surface potential decrements after aptamer interactions: 24.4 mV (9FU-AS1411), 11.7 mV (AS1411), and 2.5 mV (CRO), correlating with their binding strengths. These findings quantitatively rank aptamer affinity as 9FU-AS1411 > AS1411 >> CRO, supported by molecular-level mechanistic insights into electrostatic and structural interactions. This work pioneers high-resolution spatial mapping of nucleolin-aptamer interactions, offering novel methodologies for studying protein-aptamer binding kinetics and electrical properties at unprecedented precision (0.1 mV resolution). The approaches established here not only advance nucleolin-targeted cancer therapy but also provide a framework for investigating other protein-aptamer systems in biomedical research.

IntroductionTumor necrosis factor-α (TNF-α) is considered a potential therapeutic strategy for cancers, as it exacerbates calcium influx through voltage-gated calcium channels (VGCCs), thereby inducing apoptosis. However, the mechanisms underlying TNF-α's effects at the single-molecule level remain unclear.MethodsThis study employed multiple modes of Atomic Force Microscopy (AFM) to investigate the impact of TNF-α on breast cancer cells. The measurements were performed with nanometer spatial resolution, picoNewton force sensitivity, picoAmpere current precision, and 0.1 mV surface potential accuracy.ResultsThe results revealed that TNF-α treatment significantly increased the density and aggregation of VGCCs on the cell membrane while enhancing their channel activity. Concurrently, the electrical conductivity and surface potential of the membrane were elevated, collectively promoting exacerbated calcium influx.DiscussionThese findings elucidate the mechanisms by which TNF-α modulates VGCC distribution and electrophysiological properties to amplify calcium signaling, ultimately triggering apoptosis. This study provides unprecedented insights into TNF-α-induced calcium dysregulation in cancer cells at the single-molecule level, offering a novel approach for investigating apoptosis and advancing targeted therapies for breast cancer and other malignancies.

Breast cancer continues to present a major clinical hurdle, largely attributable to its aggressive metastatic behavior and the suboptimal efficacy of standard chemotherapeutic regimens. Cisplatin (CDDP) is a representative platinum drug in the treatment of breast cancer, however, its therapeutic application is often constrained by systemic toxicity and the frequent onset of chemoresistance. Here, we introduce a novel charge-adaptive nanoprodrug system, referred to as PP@, engineered to respond to tumor-specific conditions. This platform was constructed by conjugating ibuprofen and polyethylene glycol (PEG) to the hydrophobic and hydrophilic termini of an amphiphilic dendrimer, respectively, enabling the formation of uniform and stable nanostructures through spontaneous self-assembly. Importantly, PP@ undergoes charge reversal in response to acidic pH and elevated glutathione levels (GSH), facilitating deeper tumor penetration. Cisplatin was subsequently encapsulated within the nanoprodrug to yield the PP@-based CDDP nanoformulation (PP@CDDP). The physicochemical properties and therapeutic performance of PP@CDDP were systematically evaluated. The results demonstrated that PP@CDDP significantly improves cellular uptake, suppresses drug efflux, and reduces intracellular GSH levels, collectively contributing to prolonged drug retention at the tumor site. In vivo studies further confirmed that PP@CDDP significantly improved the antitumor efficacy of cisplatin, as evidenced by marked inhibition of tumor growth and metastasis, along with a favorable safety profile. These results underscore the potential of this charge-adaptive nanoprodrug platform to address key limitations of traditional cisplatin chemotherapy. The rational integration of smart material design with pharmacological strategies offers a promising pathway for improving therapeutic outcomes in cancer treatment.

Hepatocellular carcinoma (HCC) is a serious concern worldwide. The published reports showed that aberrant CD95 receptor expression plays a critical role in apoptosis in liver cancer. While curcumin has shown promise in inducing apoptosis in liver cancer cells, its direct effects on CD95 expression during this process have not been thoroughly investigated. This study aims to quantitatively assess the expression of the CD95 receptor in HepG2 cells treated with different concentrations of curcumin using techniques such as fluorescence staining, single-molecule force spectroscopy (SMFS), and single-molecule recognition imaging (SMRI). Fluorescence staining results indicate a significant increase in CD95 expression following curcumin treatment. For the first time, SMFS and SMRI techniques were used to directly reveal the binding sites of CD95 on the cell membrane, with the number of binding sites increasing as the curcumin concentration increased. Additionally, the binding force between an antibody-modified probe and CD95 was strengthened with curcumin treatment, suggesting that curcumin enhances both the quantity and affinity of CD95 binding sites. This study provides new insights into curcumin-induced CD95-mediated apoptosis in liver cancer cells and highlights the potential of AFM techniques for investigating drug mechanisms. Overall, these findings may inform innovative therapeutic strategies for liver cancer and improve drug design processes.

Hepatocellular carcinoma (HCC) is a highly prevalent cancer with a significant impact on human health. Curcumin, a natural compound, induces cytoskeletal changes in liver cancer cells and modifies the distribution of lipids, proteins, and polysaccharides on plasma membranes, affecting their mechanical and electrical properties. In this study, we used nanomechanical indentation techniques and Kelvin probe force microscopy (KPFM) based on atomic force microscopy (AFM) to investigate the changes in surface nanomechanical and electrical properties of nuclear and cytoplasmic regions of HepG2 cells in response to increasing curcumin concentrations. CCK-8 assays and flow cytometry results demonstrated time- and concentration-dependent inhibition of HepG2 cell proliferation by curcumin. Increasing curcumin concentration led to an initial increase and then decrease in the mechanical properties of nuclear and cytoplasmic regions of HepG2 cells, represented by the Young's modulus (E), as observed through nanoindentation. KPFM measurements indicated decreasing trends in both cell surface potential and height. Fluorescence microscopy results indicated a positive correlation between curcumin concentration and phosphatidylserine translocation from the inner to the outer membrane, which influenced the electrical properties of HepG2 cells. This study provides valuable insights into curcumin's mechanisms against cancer cells and aids nanoscale evaluation of therapeutic efficacy and drug screening.

Nucleolin is overexpressed on the surface of pancreatic cancer cells and are regarded as the remarkable therapeutic target. Aptamers are capable of binding the external domain of nucleolin on the cell surface with high affinity and specificity. But nucleolin has not been localized on pancreatic cancer cells at very high spatial resolution, and the interactions between nucleolin and aptamers have not been investigated at very high force resolution level. In this work, nucleolin was localized on pancreatic cancer and normal cells by aptamers (9FU-AS1411-NH2, AS1411-NH2 and CRO-NH2) in Single Molecule Recognition Imaging mode of Atomic Force Microscopy. There are plenty of nucleolin on the surfaces of pancreatic cancer cells (area percentage about 5%), while there are little nucleolin on the surfaces of normal cells. The interactions between three types of aptamers and nucleolins on the surfaces of pancreatic cancer cells were investigated by Single Molecule Force Spectroscopy. The unbinding forces of nucleolins-(9FU-AS1411-NH2) are larger than nucleolins-(AS1411-NH2). The dissociation activation energy on nucleolin-(9FU-AS1411-NH2) is higher than nucleolin-(AS1411-NH2), which indicates that the former complex is more stable and harder to dissociate than the later complex. There are no unbinding forces between nucleolin and CRO-NH2. All these demonstrate that nucleolin was localized on pancreatic cancer and normal cells at single molecule level quantitatively, and the interactions (unbinding forces and kinetics) between nucleolin and aptamers were studied at picoNewton level. The approaches and results of this work will pave new ways in the investigations of nucleolin and aptamers, and will also be useful in the studies on other proteins and their corresponding aptamers.

A gallium nitride (GaN) semiconductor is one of the most promising materials integrated into biomedical devices to play the roles of connecting, monitoring, and manipulating the activity of biological components, due to its excellent photoelectric properties, chemical stability, and biocompatibility. In this work, it was found that the photogenerated free charge carriers of the GaN substrate, as an exogenous stimulus, served to promote neural stem cells (NSCs) to differentiate into neurons. This was observed through the systematic investigation of the effect of the persistent photoconductivity (PPC) of GaN on the differentiation of primary NSCs from the embryonic rat cerebral cortex. NSCs were directly cultured on the GaN surface with and without ultraviolet (UV) irradiation, with a control sample consisting of tissue culture polystyrene (TCPS) in the presence of fetal bovine serum (FBS) medium. Through optical microscopy, the morphology showed a greater number of neurons with the branching structures of axons and dendrites on GaN with UV irradiation. The immunocytochemical results demonstrated that GaN with UV irradiation could promote the NSCs to differentiate into neurons. Western blot analysis showed that GaN with UV irradiation significantly upregulated the expression of two neuron-related markers, βIII-tubulin (Tuj-1) and microtubule-associated protein 2 (MAP-2), suggesting that neurite formation and the proliferation of NSCs during differentiation were enhanced by GaN with UV irradiation. Finally, the results of the Kelvin probe force microscope (KPFM) experiments showed that the NSCs cultured on GaN with UV irradiation displayed about 50 mV higher potential than those cultured on GaN without irradiation. The increase in cell membrane potential may have been due to the larger number of photogenerated free charges on the GaN surface with UV irradiation. These results could benefit topical research and the application of GaN as a biomedical material integrated into neural interface systems or other bioelectronic devices.

Calcium channel blockers (CCB) of astrocytes can blockade the calcium ions entry through the voltage gated calcium channels (VGCC), and is widely used in the diseases related with VGCC of astrocytes. But many aspects of the interaction mechanisms between the CCB and VGCC of astrocytes still remain unclear due to the limited resolution of the approaches. Herein the effects of the nicardipine (a type of CCB) on VGCC of astrocytes were investigated at very high spatial, force and electrical resolution by multiple modes of Atomic Force Microscopy (AFM) directly. The results reveal that after the addition of nicardipine, the recognition signals of VGCC disappeared; the specific unbinding forces vanished; the conductivity of the astrocytes decreased (the current decreased about 2.9 pA and the capacitance was doubled); the surface potential of the astrocytes reduced about 14.2 mV. The results of electrical properties investigations are consistent with the simulation experiments. The relations between these biophysical and biochemical properties of VGCC have been discussed. All these demonstrate that the interactions between nicardipine and VGCC have been studied at nanometer spatial resolution, at picoNewton force resolution and very high electrical signal resolution (pA in current, pF in capacitance and 0.1 mV in surface potential) level. The approaches are considered to be high resolution and high sensitivity, and will be helpful and useful in the further investigations of the effects of other types of CCB on ion channels, and will also be helpful in the investigations of mechanisms and therapy of ion channelopathies.

Voltage-gated sodium channels (VGSCs) are widely expressed in various types of tumor and cancer cells, and NaV1.5 is overexpressed in highly metastatic breast cancer cells. There may be positive relations between the expression levels of NaV1.5 and breast cancer recurrence and metastasis. Herein, NaV1.5 was detected and localized on the surfaces of normal and cancer breast cells by the single molecule recognition imaging (SMRI) mode of atomic force microscopy (AFM). The results reveal that NaV1.5 was irregularly distributed on the surfaces of normal and cancer breast cells. The NaV1.5 has an area percentage of 0.6% and 7.2% on normal and cancer breast cells, respectively, which indicates that there is more NaV1.5 on cancer cells than on normal cells. The specific interaction forces and binding kinetics in the NaV1.5-antibody complex system were investigated with the single molecule force spectroscopy (SMFS) mode of AFM, indicating that the stability of the NaV1.5-antibody on normal breast cells is higher than that on cancer breast cells. All these results will be useful to study the interactions of other ion channel-antibody systems, and will also be useful to understand the role of sodium channels in tumor metastasis and invasion.

Glucose sensing based on graphene conductance is investigated by the probe station and COMSOL Multiphysics. The current–voltage ( I–V) results from the probe station show that there is a negative linear relationship between graphene conductance and glucose concentration. Using this relationship as the standard curve, the concentration of glucose on graphene can be obtained by measuring the conductance of graphene. The electrical behaviors of different glucose molecules on graphene simulated by COMSOL Multiphysics Toolbox indicate a scattering effect of glucose molecules on current and a more rapidly linear potential drop in the glucose region calculated from the equipotential line distribution, which is consistent with the experimental results. These results provide a new research perspective and method for the sensing of glucose and other biological substances.

In recent years, semiconductors have aroused great interest in connecting, observing and influ-encing the behavior of biological elements, and it is possible to use semiconductor-cell compound interfaces to discover new signal transduction in the biological field. Among them, III-V nitride semiconductors, represented by gallium nitride (GaN), are used as substrates to form semiconductor-biology interfaces with cells, providing a platform for studying the effects of semiconductors on cell behavior. The interfaces between GaN substrate and cells play an important role in detecting and manipulating cell behaviors and provide a new opportunity for studying cell behavior and developing diagnostic systems. Hence, it is necessary to understand how the properties of the GaN substrate directly influence the behavior of biological tissues, and to create editable biological interfaces according to the needs. This paper reviews the synergism between GaN semiconductors and biological cells. The electrical properties, persistent photo-conductivity (PPC), nanostructures, and chemical functionalization of GaN on the promotion of cell behaviors, such as growth, adhesion, differentiation, and signal transduction, are emphati-cally introduced. The purpose of this study is to provide guidance to explore the detection and regulation methods of cell behavior based on semiconductors and promote the application of them in the field of bioelectronics, such as biochips, biosensors, and implantable systems.

Breast cancer is one of the most commonly diagnosed malignant cancers that threatens the health of women severely. The pathogenesis has not been revealed exhaustively due to the complex mechanisms. Evidences suggest that electrical conductivity properties play critical roles in cellular functions and activities. But the roles of electrical conductivity in pathogenesis of breast cancer cells have not been studied clearly at the nanometer level yet. In the present work, the electrical conductivity and electron transport of two normal and one cancer breast cell lines were investigated and compared at nanometer spatial level and picoampere current level by Conductive Atomic Force Microscopy (CAFM). The cell bodies of normal and cancer breast cells show the typical capacitor behaviors with little conductivity capability for electricity. The capacitance of cell bodies of the cancer breast cells is less than the normal breast cells. The conductivity of the processes of normal and cancer breast cells has also been investigated. The processes of the normal breast cells also exhibit the capacitor behavior. While the processes of the breast cells are electrically conductive along micrometer length scales, and show the semiconductor like conductive characteristics with Schottky barrier of 0.8391 V. All these demonstrate that the electrical conductivity of the cancer breast cells is better than the normal breast cells. This work will be helpful in the further investigations of electrical conductivity of normal and cancer cells at nanometer level, and will also pave new way in the distinguishing the cancer cells and tissues from the normal cells and tissues.

The interfacial mass transfer rate of a target has a significant impact on the sensing performance. The surface reaction forms a concentration gradient perpendicular to the surface, wherein a slow mass transfer process decreases the interfacial reaction rate. In this work, we self-assembled gold nanoparticles (AuNPs) in the gap of a SiO2 opal array to form a AuNP-bridge array. The diffusion paths of vertical permeability and a microvortex effect provided by the AuNP-bridge array synergistically improved the target mass transfer efficiency. As a proof of concept, we used DNA hybridization efficiency as a research model, and the surface-enhanced Raman spectroscopy (SERS) signal acted as a readout index. The experimental verification and theoretical simulation show that the AuNP-bridge array exhibited rapid mass transfer and high sensitivity. The DNA hybridization efficiency of the AuNP-bridge array was 15-fold higher than that of the AuNP-planar array. We believe that AuNP-bridge arrays can be potentially applied for screening drug candidates, genetic variations, and disease biomarkers.

Rhodopsin-like G protein-coupled receptors (GPCRs), widely distributed in microorganisms, invertebrates, and vertebrates, are the largest class in GPCRs, and are involved in many important physiological and pathological processes, including the photosensitivity, regulation of behavior and emotion, and so on. Atomic force microscopy (AFM) is a powerful and multifunctional toolkit in bionanotechnology, as it can image the morphology of membrane proteins at subnanometer spatial resolution and detect forces related with membrane proteins down to piconewton level by single-molecule force spectroscopy (SMFS) mode under physiological conditions. Herein, the achievements of AFM in the study of rhodopsin-like GPCRs, including observing the high-resolution topography and structural changes, revealing the interaction forces, binding kinetics, and mechanical properties (such as modulus), are reviewed and summarized. Finally, the challenges, outlook, and prospects of AFM in the study of rhodopsin-like GPCRs are discussed.

目的:利用石墨烯/氮化镓肖特基异质结构建一种新型的葡萄糖传感器,并对其灵敏度、线性检测范围、最低检出限和选择性等传感特性进行深入研究.方法:通过转移工艺将单层石墨烯覆盖于氮化镓单晶表面形成肖特基异质结构,将不同浓度葡萄糖溶液滴加到石墨烯表面并干燥后,利用探针台测量葡萄糖/石墨烯/氮化镓体系的电流-电压曲线,并获得开启电压,从而建立葡萄糖浓度和开启电压的对应关系.基于该对应关系可拟合得到标准曲线,并可计算出传感器的灵敏度、线性检测范围和最低检出限等传感参数,进一步将刀豆蛋白A分子修饰在石墨烯表面,实现石墨烯/氮化镓肖特基异质结对于复杂体系中葡萄糖浓度的选择性检测.结果:该新型葡萄糖传感器的标准曲线为U=0.22363+1.89968×10-4C、灵敏度为189.968 V·M-1、最低检出限为0.330μM、线性检测范围为1～1000μM、7次重复实验的相对标准差为2.89％、15天前后实验的相对标准差为0.03‰、刀豆蛋白A分子修饰样品表面后各干扰物质的影响程度均低于1.50％.结论:石墨烯/氮化镓肖特基异质结传感器是一种基于开启电压这一新型传感特性的葡萄糖传感器,其结构简单,易于制备,耐磨损腐蚀,性能优异,具有一定的应用前景.

The X-ray irradiation of gold salt aqueous solutions in the synthesis of gold nanoparticles (AuNPs) in the absence of any reducing agent or stabilizer is presented. The size, dispersion, number of particles, yield and morphology evolution during the radiolytic formation of AuNPs were followed simultaneously using in situ small-angle X-ray scattering. This study provides an insight into the overall kinetics and formation mechanisms at the initial stage of AuNP synthesis without reductants and stabilizers. The pH-dependent speciation of aqueous HAuCl4 and its influence on the synthesis, structure and properties of AuNPs were observed. The result sheds light on the key parameters required to obtain stable monomodal particles and the influence of the surface charge and reactivity of the chemical solution on the final particle size and shape.