CaMKII: a central molecular organizer of synaptic plasticity, learning and memory.

Calcium-calmodulin (CaM)-dependent protein kinase II (CaMKII) is the most abundant protein in excitatory synapses and is central to synaptic plasticity, learning and memory. It is activated by intracellular increases in calcium ion levels and triggers molecular processes necessary for synaptic plasticity. CaMKII phosphorylates numerous synaptic proteins, thereby regulating their structure and functions. This leads to molecular events crucial for synaptic plasticity, such as receptor trafficking, localization and activity; actin cytoskeletal dynamics; translation; and even transcription through synapse-nucleus shuttling. Several new tools affording increasingly greater spatiotemporal resolution have revealed the link between CaMKII activity and downstream signalling processes in dendritic spines during synaptic and behavioural plasticity. These technologies have provided insights into the function of CaMKII in learning and memory.

Biochemical Activity Architectures Visualized-Using Genetically Encoded Fluorescent Biosensors to Map the Spatial Boundaries of Signaling Compartments.

All biological processes arise through the coordinated actions of biochemical pathways. How such functional diversity is achieved by a finite cast of molecular players remains a central mystery in biology. Spatial compartmentation-the idea that biochemical activities are organized around discrete spatial domains within cells-was first proposed nearly 40 years ago and has become firmly rooted in our understanding of how biochemical pathways are regulated to ensure specificity. However, directly interrogating spatial compartmentation and its mechanistic origins has only really become possible in the last 20 or so years, following technological advances such as the development of genetically encoded fluorescent biosensors. These powerful molecular tools permit a direct, real-time visualization of dynamic biochemical processes in native biological contexts, and they are essential for probing the spatial regulation of biochemical activities. In this Account, we review our lab's efforts in developing and using biosensors to map the spatial compartmentation of intracellular signaling pathways and illuminate key mechanisms that establish the boundaries of an intricate biochemical activity architecture. We first discuss the role of regulatory fences, wherein the dynamic activation and deactivation of diffusible messengers produce diverse signaling compartments. For example, we used biosensors for the Ca2+ effector calmodulin and its downstream target calcineurin to reveal a spatial gradient of calmodulin that controls the temporal dynamics of calcineurin signaling. Our studies using cyclic adenosine monophosphate (cAMP) biosensors have similarly elucidated fenced cAMP domains generated by competing production and degradation pathways, ranging in size from cell-spanning gradients to nanoscale hotspots. Second, we describe the role played by intracellular membranes in creating unique signaling platforms with distinctive pathway regulation, as revealed through studies using subcellularly targeted fluorescent biosensors. Using biosensors to visualize subcellular extracellular response kinase (ERK) pathway activity, for example, led us to discover a local signaling circuit that mediates distinct plasma membrane ERK dynamics versus global ERK signaling. Similarly, our work developing biosensors to monitor the subcellular mechanistic target of rapamycin complex 1 (mTORC1) signaling allowed us to not only clarify the presence of mTORC1 activity in the nucleus but also identify a novel mechanism governing the activation of mTORC1 in this location. Finally, we detail how molecular assemblies enable the precise spatial tuning of biochemical activity, through investigations enabled by cutting-edge advances in biosensor design. We recently identified liquid-liquid phase separation as a major factor in cAMP compartmentation aided by a new strategy for targeting biosensors to endogenously expressed proteins via genome editing, for instance, and have also been able to directly visualize nanometer-scale protein kinase signalosomes using an entirely new class of biosensors specifically developed for the dynamic super-resolution imaging of live-cell biochemical activities. Our work provides key insights into the molecular logic of spatially regulated signaling and lays the foundation for a broader exploration of biochemical activity architectures across multiple spatial scales.

Strategy to Immobilize Peptide Probe Selected through In Vitro Ribosome Display for Electrochemical Aptasensor Application.

In this study, we demonstrated an electrochemical aptasensor for calmodulin (CaM) detection and the peptide sequence (YWDKIKDFIGG) is obtained from in vitro ribosome display selection. To immobilize this peptide probe on the electrode surface, cystine was incorporated at the end of this peptide sequence. After a maleimide-functionalized poly(3,4-ethylenedioxythiophene), poly(EODT-MI), film was electropolymerized on the electrode, the peptide probe was immobilized through thiol-ene conjugation with the cystine end. Four peptides with different linkers were used for the binding test of bovine serum albumin and CaM using a quartz crystal microbalance. The zwitterionic linker EKEKEKEKEKEK provided good antifouling properties and the highest CaM binding. Furthermore, the immobilization of the peptide with this zwitterionic linker resulted in a minimal increase in the electrochemical impedance. By immobilizing the peptide with the selected zwitterionic linker, we successfully demonstrated an electrochemical aptasensor with a linear detection range for CaM from 0.01 to 10 mg/L and a detection limit of 0.001 mg/L.

In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain.

High-resolution optical imaging is critical to understanding brain function. We demonstrate that three-photon microscopy at 1,300-nm excitation enables functional imaging of GCaMP6s-labeled neurons beyond the depth limit of two-photon microscopy. We record spontaneous activity from up to 150 neurons in the hippocampal stratum pyramidale at ∼1-mm depth within an intact mouse brain. Our method creates opportunities for noninvasive recording of neuronal activity with high spatial and temporal resolution deep within scattering brain tissues.

In Situ Living Cell Protein Analysis by Single-Step Mass Spectrometry.

In situ living cell protein analysis would enable the structural identification and functional interrogation of intracellular proteins in native cellular environments. Previously, we have presented an in situ mass spectrometry (MS) strategy to identify protein and protein/metal ion complex with relatively small molecular weight ( Anal. Chem. 2016, 88, 10860-10866). However, it is still challenging to directly identify larger proteins and protein/ligand complexes in cell, due to numerous nonspecific bindings of ligands, solvents, and other cellular constituents. Here we present a versatile single-step mass spectrometric strategy, "in-cell" mass spectrometry ("in-cell" MS), for in situ protein identification and dynamic protein-ligand interaction monitoring directly from living cells. "In-cell" MS combined all-ion-fragmentation mode with our previous method; thus, on a high-resolution MS instrument, we can greatly improve the signal/noise ratio of the larger proteins and protein/ligand complexes. Meanwhile, we also achieved a much wider mass range for protein complex and detection of 17 proteins with molecular weight ranging from 4 to 44 kDa. In addition, "in-cell" MS could also monitor dynamic protein interactions in living cells. Calcium-regulated calmodulin-melittin interaction was tested to demonstrate the proof of concept. "In-cell" MS provides an alternative for in situ analysis of living cells, which might contribute to rapid protein analysis and quality control in biochemistry laboratories, protein engineering, and even protein industry.

Tracking and localization of calmodulin in live cells.

The calcium signaling protein calmodulin (CaM) interacts with many target proteins inside the cell to regulate a wide range of biological signals. CaM's availability to propagate signals depends on its mobility, which may be regulated by interactions with multiple target proteins. We detected single molecules of CaM labeled with a fluorescent dye and injected into living HEK 293 cells, and we used high-speed, wide-field, single-molecule imaging to track single CaM molecules. Single-molecule trajectories were analyzed to characterize the motions of individual CaM molecules. Single-molecule localization resolved CaM positions with a position accuracy of <100nm, permitting sub-diffraction imaging of features with localized CaM that form in response to increased free Ca(2+). Single-molecule tracking demonstrated the presence of a wide range of mobilities of individual calmodulin molecules in a cell, with diffusion coefficients ranging from <0.01µm(2)s(-1) to ~5µm(2) s(-1), whereas analysis by spatio-temporal image correlation spectroscopy revealed faster-moving components with diffusion coefficients of >10µm(2)s(-1). For molecules confined to small regions of the cell, super-resolved images of presumed signaling complexes were recovered. Individual trajectories were classified as normal diffusion, confined diffusion, or directed motion, and could suggest how the individual CaM molecules were bound in the cell. The results show that interactions of CaM with target proteins result in decreased translational mobilities of a significant fraction of CaM molecules inside cells. The work presented here illustrates methods that can characterize location, mobilities, and the availability of signaling molecules in live cells.

A cryptophane-based "turn-on" 129Xe NMR biosensor for monitoring calmodulin.

We present the first cryptophane-based "turn-on" 129Xe NMR biosensor, employing a peptide-functionalized cryptophane to monitor the activation of calmodulin (CaM) protein in solution. In the absence of CaM binding, interaction between the peptide and cryptophane completely suppresses the hyperpolarized 129Xe-cryptophane NMR signal. Biosensor binding to Ca2+-activated CaM produces the expected 129Xe-cryptophane NMR signal.

Cloning and Stress-Induced Expression Analysis of Calmodulin in the Antarctic Alga Chlamydomonas sp. ICE-L.

Calmodulin (CaM) is a Ca2+-binding protein that plays a role in several Ca2+ signaling pathways, which dynamically regulates the activities of hundreds of proteins. The ice alga Chlamydomonas sp. ICE-L, which has the ability to adapt to extreme polar conditions, is a crucial primary producer in Antarctic ecosystem. This study hypothesized that Cam helps the ICE-L to adapt to the fluctuating conditions in the polar environment. It first verified the overall length of Cam, through RT-PCR and RACE-PCR, based on partial Cam transcriptome library of ICE-L. Then, the nucleotide and predicted amino acid sequences were, respectively, analyzed by various bioinformatics approaches to gain more insights into the computed physicochemical properties of the CaM. Potential involvements of Cam in responding to certain stimuli (i.e., UVB radiation, high salinity, and temperature) were investigated by differential expression, measuring its transcription levels by means of quantitative RT-PCR. Results showed that CaM was indeed inducible and regulated by high UVB radiation, high salinity, and nonoptimal temperature conditions. Different conditions had different expression tendencies, which provided an important basis for investigating the adaptation mechanism of Cam in ICE-L.

Xilmass: A New Approach toward the Identification of Cross-Linked Peptides.

Chemical cross-linking coupled with mass spectrometry plays an important role in unravelling protein interactions, especially weak and transient ones. Moreover, cross-linking complements several structural determination approaches such as cryo-EM. Although several computational approaches are available for the annotation of spectra obtained from cross-linked peptides, there remains room for improvement. Here, we present Xilmass, a novel algorithm to identify cross-linked peptides that introduces two new concepts: (i) the cross-linked peptides are represented in the search database such that the cross-linking sites are explicitly encoded, and (ii) the scoring function derived from the Andromeda algorithm was adapted to score against a theoretical tandem mass spectrometry (MS/MS) spectrum that contains the peaks from all possible fragment ions of a cross-linked peptide pair. The performance of Xilmass was evaluated against the recently published Kojak and the popular pLink algorithms on a calmodulin-plectin complex data set, as well as three additional, published data sets. The results show that Xilmass typically had the highest number of identified distinct cross-linked sites and also the highest number of predicted cross-linked sites.

Binding States of Protein-Metal Complexes in Cells.

The identification of endogenous proteins as well as their binding to metal ions in living cells is determined by combining pulsed electrophoretic separations with nanoelectrospray ionization followed by mass spectrometric detection. This approach avoids problems resulting from the complicated cellular environment. In this manner, we demonstrate the rapid identification (300 ms or less) of intact proteins from living E. coli cells including the complexation of calmodulin with calcium ion. The latter showed different binding states from those observed in in vitro studies. These observations also reveal in vitro measurements do not necessarily represent the actual situation in living cells. We conclude that the attempted in situ measurement of intracellular proteins with minimal sampling processes should be preferred.

Mapping protein structural changes by quantitative cross-linking.

Chemical cross-linking is a promising technology for protein tertiary structure determination. Though the data has low spatial resolution, it is possible to obtain it at physiological conditions on proteins that are not amenable to standard high resolution techniques such as X-ray, NMR analysis and cryo-EM. Here we demonstrate the utilization of isotopically labeled chemical cross-linking to visualize protein conformation rearrangements. Since calmodulin exists in two distinct conformations (calcium-free and calcium-containing forms), we selected this protein for testing the potential and the limits of a new technique. After cross-linking of both calmodulin forms, the calcium-free and calcium-containing forms were mixed together and digested under different conditions and the products of proteolysis were monitored using high resolution mass spectrometry. Finally, the ratios of heavy/light cross-links were calculated by mMass open source platform.

Extending the cross-linking/mass spectrometry strategy: Facile incorporation of photo-activatable amino acids into the model protein calmodulin in Escherichia coli cells.

Photo-induced cross-linking is a highly promising technique to investigate protein conformations and protein-protein interactions in their natural cellular environment. One strategy relies on the non-directed incorporation of diazirine-containing photo-activatable amino acids into proteins and a subsequent cross-link formation induced by UV-A irradiation. The advantage of this photo-cross-linking strategy is that it is not restricted to lysine residues and that hydrophobic regions in proteins can also be targeted, which is advantageous for investigating membrane proteins. Here, we present a simplified protocol that relies on the use of mineral salts medium without any special requirements for the incorporation of photo-methionines into proteins in Escherichia coli cells. The possibility to perform these experiments in E. coli is especially valuable as it is the major system for recombinant protein production. The method is exemplified for the Ca(2+) regulating protein calmodulin containing nine methionines, which were found to be replaced by their photo-activatable analogues. Our protocol allows the facile and stochastic incorporation of photo-methionines as the basis for conducting photo-cross-linking experiments in E. coli in an efficient manner.

Enzyme-inhibitor-like tuning of Ca(2+) channel connectivity with calmodulin.

Ca(2+) channels and calmodulin (CaM) are two prominent signalling hubs that synergistically affect functions as diverse as cardiac excitability, synaptic plasticity and gene transcription. It is therefore fitting that these hubs are in some sense coordinated, as the opening of Ca(V)1-2 Ca(2+) channels are regulated by a single CaM constitutively complexed with channels. The Ca(2+)-free form of CaM (apoCaM) is already pre-associated with the isoleucine-glutamine (IQ) domain on the channel carboxy terminus, and subsequent Ca(2+) binding to this 'resident' CaM drives conformational changes that then trigger regulation of channel opening. Another potential avenue for channel-CaM coordination could arise from the absence of Ca(2+) regulation in channels lacking a pre-associated CaM. Natural fluctuations in CaM concentrations might then influence the fraction of regulable channels and, thereby, the overall strength of Ca(2+) feedback. However, the prevailing view has been that the ultrastrong affinity of channels for apoCaM ensures their saturation with CaM, yielding a significant form of concentration independence between Ca(2+) channels and CaM. Here we show that significant exceptions to this autonomy exist, by combining electrophysiology (to characterize channel regulation) with optical fluorescence resonance energy transfer (FRET) sensor determination of free-apoCaM concentration in live cells. This approach translates quantitative CaM biochemistry from the traditional test-tube context into the realm of functioning holochannels within intact cells. From this perspective, we find that long splice forms of Ca(V)1.3 and Ca(V)1.4 channels include a distal carboxy tail that resembles an enzyme competitive inhibitor that retunes channel affinity for apoCaM such that natural CaM variations affect the strength of Ca(2+) feedback modulation. Given the ubiquity of these channels, the connection between ambient CaM levels and Ca(2+) entry through channels is broadly significant for Ca(2+) homeostasis. Strategies such as ours promise key advances for the in situ analysis of signalling molecules resistant to in vitro reconstitution, such as Ca(2+) channels.

Salt gradient enhanced sensitivity in nanopores for intracellular calcium ion detection.

Intracellular calcium ion detection is of great significance for understanding the cell metabolism and signaling pathways. Most of the current ionic sensors either face the size issue or sensitivity limit for the intracellular solution with high background ion concentrations. In this paper, we proposed a calmodulin (CaM) functionalized nanopore for sensitive and selective Ca2+ detection inside living cells. A salt gradient was created when the nanopore sensor filled with a low concentration electrolyte was in contact with a high background concentration solution, which enhanced the surface charge-based detection sensitivity. The nanopore sensor showed a 10 × sensitivity enhancement by application of a 100-fold salt gradient, and a detection limit of sub nM. The sensor had a wide detection range from 1 nM to 1 mM, and allowed for quick calcium ion quantification in a few seconds. The sensor was demonstrated for intracellular Ca2+ detection in A549 cells in response to ionomycin.

Molecular identification, immunolocalization, and characterization of Clonorchis sinensis calmodulin.

One cDNA clone (Cs18h09) encoding Clonorchis sinensis calmodulin (CsCaM) was isolated from our adult cDNA plasmid library. The open reading frame of CsCaM contains 450 bp which encodes 149 amino acids. CsCaM protein comprises four calcium-binding EF-hand motifs. The amino acid sequence of CsCaM shares very high homology with other species. Quantitative RT-polymerase chain reaction (PCR) revealed that CsCaM mRNA was constitutively transcribed in development cycle stages of the parasite, including adult worm, metacercaria, excysted metacercaria, and egg. In addition, recombinant CsCaM (rCsCaM) was expressed as a soluble protein and anti-rCsCaM rat serum could detect CsCaM in the C. sinensis somatic extracts but not in the C. sinensis excretory-secretory products (ESPs). Moreover, immunolocalization assay showed that CsCaM was located in tegument, intestine, pharynx, and eggs. Furthermore, rCsCaM was found to bind calcium ion (Ca2+) and magnesium (Mg2+) in electrophoretic mobility shift assay. Ca2+ binding increased the ability of rCsCaM to bind the hydrophobic fluorescent probe 8-anilinonaphthalene-1-sulphonate, causing a blue shift in the fluorescence emission from 540 to 515 nm with an excitation wavelength of 380 nm and substantial increase in fluorescence intensity but not Mg2+. Collectively, here we showed the basic characterization of CsCaM and inferred that CsCaM could be a Ca2+ sensor protein, and CsCaM may possibly participate in growth and development of adult worm and egg of C. sinensis through binding Ca2+.

Towards the discovery of goat meat quality biomarkers using label-free proteomics.

This study aimed to identify for the first time protein biomarkers of meat quality traits from Longissimus thoracis (LT) muscle of goats (Capra hircus). Male goats of similar age and weight reared under extensive rearing conditions were used to relate the LT muscle proteome with multiple meat quality traits. The early post-mortem muscle proteome analyzed using label-free proteomics was compared among three texture clusters built using hierarchical clustering analysis. Twenty-five proteins were differentially abundant and their mining using bioinformatics revealed three major biological pathways to be involved: 10 muscle structure proteins (MYL1, MYL4, MYLPF, MYL6B, MYH1, MYH2, ACTA1, ACTBL2, FHL1 and MYOZ1); 6 energy metabolism proteins (ALDOA, PGAM2, ATP5F1A, GAPDH, PGM1 and ATP5IF1), and two heat shock proteins: HSPB1 (small) and HSPA8 (large). Seven other miscellaneous proteins belonging to pathways such as regulation, proteolysis, apoptosis, transport and binding, tRNA processing or calmodulin-binding were further identified to play a role in the variability of goat meat quality. The differentially abundant proteins were correlated with the goat meat quality traits in addition to multivariate regression models built to propose the first regression equations of each quality trait. This study is the first to highlight in a multi-trait quality comparison the early post-mortem changes in the goat LT muscle proteome. It also evidenced the mechanisms underpinning the development of several quality traits of interest in goat meat production along the major biochemical pathways at interplay. SIGNIFICANCE: The discovery of protein biomarkers in the field of meat research is an emerging topic. In the case of goat meat quality, very few studies using proteomics have been conducted with the aim of proposing biomarkers. Therefore, this study is the first to quest for biomarkers of goat meat quality using label-free shotgun proteomics with a focus on multiple quality traits. We identified the molecular signatures underlying goat meat texture variation, which were found to belong to muscle structure and related proteins, energy metabolism and heat shock proteins along with other proteins involved in regulation, proteolysis, apoptosis, transport and binding, tRNA processing or calmodulin-binding. We further evaluated the potential of the candidate biomarkers to explain meat quality using the differentially abundant proteins by means of correlation and regression analyses. The results allowed the explanation of the variation in multiple traits such as pH, color, water-holding capacity, drip and cook losses traits and texture.

Mass spectrometry-based sequencing of protein C-terminal peptide using α-carboxyl group-specific derivatization and COOH capturing.

An approach to mass spectrometry (MS)-based sequence analysis of selectively enriched C-terminal peptide from protein is described. This approach employs a combination of the specific derivatization of α-carboxyl group (α-COOH), enzymatic proteolysis using endoproteinase GluC, and enrichment of C-terminal peptide through the use of COOH-capturing material. Highly selective derivatization of α-COOH was achieved by a combination of specific activation of α-COOH through oxazolone chemistry and amidation using 3-aminopropyltris-(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-propylamine). This amine component was used to simplify fragmentation in tandem mass spectrometry (MS/MS) measurement, which facilitated manual sequence interpretation. The peptides produced after GluC digestion were then treated with a COOH scavenger to enrich the C-terminal peptide that is only devoid of COOH groups, and the obtained C-terminal peptide was readily sequenced by matrix-assisted laser desorption/ionization (MALDI)-MS/MS due to the TMPP mass tag.

Calmodulin-like protein from Entamoeba histolytica: solution structure and calcium-binding properties of a partially folded protein.

The mechanism of Ca(2+)-signaling in the protozoan parasite Entamoeba histolytica is yet to be understood as many of the key regulators are still to be identified. E. histolytica encodes a number of multi-EF-hand Ca(2+)-binding proteins (EhCaBPs). Functionally only one of these molecules, EhCaBP1, has been characterized to date. The calmodulin-like protein from E. histolytica (abbreviated as EhCaM or EhCaBP3) is a 17.23 kDa monomeric protein that shows maximum sequence identity with heterologous calmodulins (CaMs). Though CaM activity has been biochemically shown in E. histolytica, there are no reports on the presence of a typical CaM. In an attempt to understand the structural and functional similarity of EhCaM with CaM, we have determined the three-dimensional (3D) solution structure of EhCaM using NMR. The EhCaM has a well-folded N-terminal domain and an unstructured C-terminal counterpart. Further, it sequentially binds only two calcium ions, an unusual mode of Ca(2+)-binding among the known CaBPs, notably both in the N-terminal domain of EhCaM. Further, EhCaM is present in the nucleus in addition to the cytoplasm as detected by immunofluorescence staining, unlike other EhCaBPs that are detected only in the cytoplasm. Therefore, this protein is likely to have a different function. The presence of unusual and a diverse set of CaBPs in E. histolytica suggests a distinct Ca(2+)-signaling process in E. histolytica. The results reported here help in understanding the structure-function relationship of CaBPs including their Ca(2+)-binding properties.

Mass spectrometry of laser-initiated carbene reactions for protein topographic analysis.

We report a protein labeling method using nonselective carbene reactions of sufficiently high efficiency to permit detection by mass spectrometric methods. The approach uses a diazirine-modified amino acid (l-2-amino-4,4'-azipentanoic acid, "photoleucine") as a label source, which is converted to a highly reactive carbene by pulsed laser photolysis at 355 nm. Labeling of standard proteins and peptides (CaM, Mb, M13) was achieved with yields up to 390-fold higher than previous studies using methylene. Carbene labeling is sensitive to changes in protein topography brought about by conformational change and ligand binding. The modification of apo-CaM was 45 ± 7% higher than that of holo-CaM. Modification of the CaM-M13 complex reflected a 39 ± 1% reduction in labeling for bound holo-CaM relative to free holo-CaM. Labeling yield is independent of protein concentration over approximately 2 orders of magnitude but is weakly dependent on the presence of other chromophores in a photon-limited apparatus. The current configuration required 2 min of irradiation for full reagent conversion; however, it is shown that comparable yields can be achieved with a single high-energy laser pulse (>100 mJ/pulse, <10 ns), offering a labeling method with high temporal resolution. We suggest a mechanism of labeling governed by limited carbene diffusion and the protein surface activity of the diazirine precursor. This surface activity is speculated to return a measure of selectivity relative to methylene labeling, which ultimately may be tunable.

Study on a Plasmonic Tilted Fiber Grating-Based Biosensor for Calmodulin Detection.

Tilted fiber Bragg grating, which has the advantages of both fiber Bragg grating and long-period fiber grating, has been widely studied for sensing in many fields, especially in the field of biochemistry. Calmodulin, which has a wide distribution in eukaryotes, can regulate several enzymes such as adenylate cyclase and guanylate cyclase and mediates several cellular processes such as cell proliferation and cyclic nucleotide metabolism. The abnormal levels of calmodulin in the body will result in serious effects from metabolism to nerve growth and memory. Therefore, it is important to measure the calmodulin concentration in the body. In this work, we propose and experimentally demonstrate a plasmonic tilted fiber Bragg grating-based biosensor for calmodulin detection. The biosensor was made using an 18° tilted fiber Bragg grating with a 50 nm-thick gold nanofilm coating the surface of the fiber, and transient receptor potential channels were bonded onto the surface of the gold nanofilm to serve as bio-detectors for calmodulin detection. Experimental results showed that the limit of detection using our biosensor was 0.44 nM. Furthermore, we also demonstrated that the interaction between calmodulin and transient receptor potential channels was quite weak without calcium in the solution, which agrees with the biology. Our proposed biosensor has a simple structure, is easy to manufacture, and is of small size, making it a good choice for real-time, label-free, and microliter-volume biomolecule detection.