Nanodevices for Biological and Medical Applications: Development of Single-Molecule Electrical Measurement Method

A comprehensive detection of a wide variety of diagnostic markers is required for the realization of personalized medicine. As a sensor to realize such personalized medicine, a single molecule electrical measurement method using nanodevices is currently attracting interest for its comprehensive simultaneous detection of various target markers for use in biological and medical application. Single-molecule electrical measurement using nanodevices, such as nanopore, nanogap, or nanopipette devices, has the following features:; high sensitivity, low-cost, high-throughput detection, easy-portability, low-cost availability by mass production technologies, and the possibility of integration of various functions and multiple sensors. In this review, I focus on the medical applications of single- molecule electrical measurement using nanodevices. This review provides information on the current status and future prospects of nanodevice-based single-molecule electrical measurement technology, which is making a full-scale contribution to realizing personalized medicine in the future. Future prospects include some discussion on of the current issues on the expansion of the application requirements for single-mole-cule measurement.


Introduction
Personalized medicine is known as tailor-made medical care and healthy life system for each person by providing appropriate treatment and medication. In order to realize these personalized medication systems, the development of sensing technologies, which can detect detailed personal health status and information by monitoring various medical sensors, is essential. Single-molecule electrical measurement using nanostructure integrated devices, called "nanodevice", is one of the candidates to addressing the issues in this realization of personalized medicine ( Figure 1). Compared to conventional analytical techniques, there are several advantages of single-molecule electrical measurements using nanodevices: high sensitivity, which enables early diagnosis and monitoring, the simplicity and compactness of the measurement system, which enables easy-portability in field works and research; and the possibility of the integration of various functions and multiple sensors such as separation and purification.
Nanopore based single molecule measurement is one of the most attractive single molecule measurements using nanodevices [1,2]. The detection principle of nanopore based single molecule measurement methods is the same as the Coulter counter detection principle. The principles are herein briefly described. The nanopore device has two solution chambers separated by a membrane with a single nanopore, and the chambers are filled with an electrolyte solution. When a single molecule/particle flows into the nanopore, the electrical conductivity of the electrolyte solution in the pore decreases due to In personalized medicine, non-invasive liquid biopsy (urine, blood, etc.) is expected to be used for the application of sampling. The samples include miRNAs and proteins released from the liposomes of cells, as well as the virus itself in case of infection. The target markers in these samples are electrically measured at the single molecule level using nanodevices, such as nanopores, nanogap electrodes, and nano fluid/channels. As single-molecule detection methods, electrical and optical and its combination measurements have been reported.
Nanogap based single molecule measurement is also used for nanodevices for single molecule electrical measurement [6]. The measurement principle is to measure the electrical conductivity of individual molecules by using around 1 nm gaps, which are formed by using a mechanically controllable or electrical break junction method. Sample molecules enter and translocate through the nanogap-electrodes, ensuring the facilitation of the tunneling current via the nucleotide molecules. The tunnel-current intensity is related to the molecular energy level so that signal intensity represents the electrical conductivity of individual molecules. This method enables the analysis of chemical bonds, steric structure, molecular interactions between target molecules and gold electrodes, and the polarity of sample molecules.
With the recent development of the nanodevice fabrication by semiconductor massproduction technique, the fabrication cost of the devices has rapidly decreased so that the nanodevice sensors are able to be utilized for biological/medical applications. In this review, we introduce the cutting-edge medical applications that have been made possible by using nanodevices.

Single Molecule Electrical Measurements: Biomolecule Detection
The first step for disease diagnosis and health status is the detection of biomolecules. The detection methods are roughly separated into two categories: direct sensing and indirect sensing for single molecule measurement using nanodevices.

Direct Sensing by Single Molecule Electrical Measurements
Direct sensing involves directly reading physical properties from the target molecule. Physical properties include the electronic state, the physical volume, which include the solvation shell of the molecule/ions, and structural differences, which are induced by conformational change of the molecule via inter/intra molecular interaction ( Figure 2). These direct sensing methods are applied to single-molecule sequencing for biological polymers such as DNA, RNA, and peptides. These direct sensing methods potentially contribute to the detection of rare target markers and the discovery of new target markers. Direct sensing of target molecule/particle by using nanodevices. In single molecule/particle measurement using nanodevices, an electrical signal for a single molecule is observed as a current-time profile when the molecule passes through the sensor device. In the case of nanopore devices, the intensity of decrement in ion current during passage represents the volume of the molecule (a), and in the case of nanogap devices, the signal intensity of the tunnel-current represents the electronic conductivity of the molecule (b). When a single biopolymer passes through the sensor, each unit of the polymer (nucleotide for DNA and RNA, amino acid for peptide and protein) passes through the sensor, and the electrical signal reflects the sequence of the biopolymer (c). Detection of different molecular signals occurs even for the same target molecule. If a structural change has occurred, different electrical signals reflecting the different structures would be observed (d-f). In fact, signals reflecting differences in the intramolecular hydrogen bond morphology of proteins (d:prion) and nucleic acid base chains (DNA single-strand, duplex, and triplex) have been observed. In addition, differences of shapes in the nanoparticles and viruses have also been identified by electrical signals reflecting the shape of the particles (f).

Indirect Sensing by Single Molecule Electrical Measurements
Indirect sensing involves detecting the specific molecular marker by using analytical probes, which are designed to selectively bind to the target marker and serve to amplify the electrical signal by nanodevices ( Figure 3). By using probe molecules, target marker molecules specifically form a host (probe)-guest (target) conjugate, resulting in the signal amplification of target molecules so that they are easily discriminated from the host only (probe) and no-target other molecules. In addition to signal amplification, some probe molecules work as chemical scavengers in order to purify or exclude molecules that interfere with detection other than guests. The use of these probe molecules enables the specific and selective detection of guests, resulting in an improvement of the sensing accuracy and sensitivity. Figure 3. Indirect sensing of target molecule/particle by nanodevices. In this sensing mode, a probe molecule that selectively binds to the target molecule is required. DNA aptamer (a) and complementary nucleotides (b) are used as probe molecules. In addition to nucleotide probe molecules, peptide chains and monoclonal antibodies (c) are used to measure the binding form of target molecules and particles using nanodevices, and identify the differences in electrical signals.

Nucleotide Sensing
Nucleic acid and nucleotides, such as DNA and RNA, are biologically important targets for understanding diseases and health conditions caused by genetic abnormalities and abnormal expression, as DNA is the main body of genes, and RNA is a substance that is translated from DNA during gene expression. Recently, in addition to the natural nucleobases [7][8][9][10][11][12][16][17][18][19]24], the detection of post-translational modifications has become important and interesting as epi-transcriptome research fields because these nucleotide and nucleobase modifications play roles in gene expression and suppression, which are closely related to various biological malfunction such as disease and aging. Since there are about over one hundred types of known base modifications in eukaryotic cells, some of the epi-modification, such as methylated nucleobases (e.g., methyl cytosine, methyl adenine) and oxidized guanine are detected by these nanodevice detection methods [13][14][15][20][21][22][23].

Amino Acids, Peptides and Proteins Sensing
Amino acids, peptides and proteins are also important target molecules because they perform various biological functions such as catalyzing metabolic reactions, DNA replication, signal transduction and metabolism thus abnormalities are closely related to diseases. Up to now, there have been reports on the detection of the twenty amino acids, and post-translational modifications of amino acids such as methylation, acetylation, and phosphorylation have also been detected [25][26][27][28]. Peptide and proteins, which are polymers of amino acids, are also important targets. Besides reading sequences or direct sensings by nanodevices, the indirect sensing method is often utilized for the detection of proteins such as streptavidin, maltose-binding protein (MBP), etc., by using probes such as nucleic acid aptamers that bind to the host molecules of specific target molecules [29][30][31][32][33].

Glycans, and Biocompatible Polymer (PEG) Sensing
Glycans are an important biopolymer, belong to group of compounds consisting of various sugars connected by glycosidic bonds. Glycans can bind not only to other sugars but also to proteins, lipids, and other small molecules to produce a variety of molecules. These glycoproteins and glycolipids are important biopolymers that play important physiological roles in living organisms, and their detection has attracted much attention. Such glycosides and its related molecules have been reported by single-molecule detection [34][35][36]. Besides this, biocompatible polymers have attracted interest as nanodevice modification substances. Among them, polyethylene glycol (PEG) is often used in the chemical modification of nanodevices for avoiding the non-specific sticking of protein on the devices. In the reports of single-molecule measurements, the degree of polymerization of PEGs have been detected [37][38][39].

Second Messengers, Ion Sensing
Second messengers and ions, which play an important role in the transmission of information in the body, are also important targets. Among the second messengers, the detection of cAMP and neurotransmitters such as adrenaline, a neurotransmitter are reported [41][42][43][44][45]. In these reports, direct sensing of these molecules were successfully achieved by optimizing the size of nanopores and nanogap for nanodevices because the nanodevice sensors are sensitive to the size of molecular volume, three-dimensional structure, and hydration radius of the targets. In the case of indirect sensing, probe molecules, which selectively interact with the target molecules, are utilized for detection of the target ion [46][47][48][49][50][51][52]. For instance, this method has successfully detected harmful ions such as uranium [49] and mercury ions [50]. The strategy of size optimization for nanodevices also works for detection of viruses, cells, particulate matter (PM), etc. [53][54][55].

Single-Molecule Electrical Detection Based Sequencing
Based on the sequential identification of chemical species such as nucleotides and amino acids by single molecule electrical measurements, single-molecule electrical sequencing was developed to read sequences for biopolymers of DNA, RNA, and peptides ( Figure 4) [56][57][58][59][60][61][62][63][64]. Nucleic acid sequencing [56][57][58][59][60] and peptide sequencing [61,62] methods have been proposed and recently developed. Compared to conventional sequencers, this single molecule electrical sequencer has the following features: first, this sequencing methodology does not need any amplification process, thus it can reduce the analytical time and cost of reagents for amplification, and allows the equipment to become smaller; second, this electrical sequencing methodology can read DNA information faster than the conventional optical probe based sequencing technologies as optical probe-based sequencing needs a DNA elongation reaction; third, epigenetic information can be detected as the native sample molecules are directly observed without amplification. Particularly, single-molecule DNA sequencing is increasingly utilized for on-site portable sequencing because it is cheaper and more compact than conventional DNA sequencers. Tumor-specific marker molecules in liquid biopsy samples, such as blood, urine, and saliva obtained from patients, are measured. After these samples are pretreated by purification, amplification, and chemical treatment, single-molecule measurements are performed using nanodevices to distinguish between patients and healthy individuals. There are several reports of the detection of genes and miRNAs by sequencing, and/or detection using probe molecules that selectively bind to the target.

Cancer Diagnosis
One of the most widely reported studies is cancer diagnosis. It is generally known that cancer is caused by genetic abnormalities in cells, but there are many possible causes, and the corresponding diagnostic markers are different for each of the causes. In the application of cancer diagnosis for personal medical care, it is necessary to find the cause of each cancer individually and to administer the corresponding drug appropriately. Therefore, the simultaneous detection of various diagnostic marker molecules is required.
Diagnostic marker molecules for cancer diagnosis include specific DNA gene sequences, specific genomic sequence duplications such as specific short tandem repeats (STRs), overexpression or suppression of miRNAs, CpG methylation, and tumor marker proteins and so on.
Sequencing methods by nanodevices are utilized for the detection of cancer related gene sequences such as genetic mutations and polymorphisms [63][64][65][66][67]. For example, the genetic abnormality of TP53, a specific oncogene, was detected in blood samples [67]. It is possible to perform the detection by the Illumina and Sanger sequencing method but the nanodevice based single-molecule sequencer can detect them cheaper and faster.
Indirect detection methods are utilized for the detection of cancer markers by using nucleotide probes, which are designed to selectively bind to the target cancer marker. For instance, by using a nucleic acid probe with complementary base pairs and a target miRNA, the detection of cancer-related miRNAs was successfully achieved [68][69][70][71][72][73][74][75]. In this method, a specific miRNA is detected by the probe molecule bound to the miRNA conjugates in blood. In addition, by using multi targeting probes, which can be hybridized with several types of miRNAs, multiple target miRNAs were detected simultaneously [70,71]. For no nucleotide type target markers, detection by using the nucleotide probe, which can interact with target markers, was successfully achieved [79,81]. For instance, using a nucleic acid aptamer probe, prostate-specific antigen (PSA) was detected [79]. The development of the evolutionary engineering method using nucleic acids and peptides can produce probe molecules with high selectivity so that the nanodevice detection method using probe molecules can become more accurate in the future. CpG methylation has been reported in various cancers such as colorectal cancer and lung cancer, and has attracted attention as target cancer markers. By using single-molecule detection with nanodevices, such epi-genetic detection was also reported [83].

Alzheimer's Disease, Huntington's Disease, and Prion Diseases Detection
In addition to cancer diagnoses, there have been reports on the detection of Alzheimer's disease, Huntington's disease, and prion diseases related to target markers [101][102][103][104][105][106]. These diseases are known to be induced by abnormal protein aggregation, thus the detection of the protein aggregation is the first step for the disease diagnosis. For instance, single-molecule detection method by nanodevice succeeded in detecting differences in the aggregation structure of amyloid-β [103,104] and prion [105]. Therefore, these methods are expected to contribute to the understanding of the cause of the disease, early detection, and the development of drugs.

Virus Detection
It has become an important target to diagnose the presence or absence of viral infection cheaply, quickly and accurately ( Figure 5) due to the recent COVID-19 pandemic having raised interest in on-site sequencing to monitor mutant strains. Up to now, there have been several reports on virus detection by single-molecule detection methods [53,64,[88][89][90][91][92][93][94]. They are mainly categorized into two methods. The first is the detection of viral RNA sequences, and the second is the detection of viral particle shapes. For instance, the first method successfully detected influenza A [89], HPV [64], Lhasa fever [110], and Ebola [85] by on-site sequencing with Minion. The second method is the direct detection of differences in the three-dimensional structure and shape of viruses. Using this method, various types of viruses, such as Influenza A and B, Coronavirus, Adenovirus, and Respiratory Syncytial virus [88], along with HCoV-229E, SARS-CoV, MERS-CoV, and SARS-CoV-2 [53], have been successfully identified.

Figure 5.
Virus detection by single-molecule/particle electrical detection with a nanopore device. Sample viruses are collected from liquid biopsy samples, such as nasal swabs, saliva, blood, and urine, etc. As targets, there are virus whole-particles, RNA and/or its cDNA converted from the virus RNA, and parts of the virus particles and its proteins such as spikes that constitute the virus. The presence of viruses has been detected by identifying the shape of the virus, sequencing nucleic acid base chains, detecting known target nucleic acid base sequences by complementary strand sequences, and detecting target markers such as proteins by using probe molecules that selectively bind to them.

Drug Screening and Environmental Monitoring
Single-molecule drug screening is one of the major targets due to its featuring inexpensive measurement systems and rapid evaluation of various candidate chemical substances. The detection of drugs by single-molecule detection methods has been reported [89][90][91][92][93][94]. For example, drugs such as ibuprofen [95], doxorubicin [99], and trifluridine (FTD) [100] were detected. It is expected that further integration of these single-molecule detection technologies enables comprehensive drug screening by using the Total Analysis System (TAS), which consists of various kinds of probe molecules immobilized sensor array.
In the application of environmental monitoring, these single-molecule measurements by nanodevices are expected to provide stable, long-term measurements of a wide variety of target molecules with low costs. The application of environmental monitoring includes the constant monitoring of pollutants in the air, aqueous solutions, and soil, such as toxic and hazardous substances. Among them, metagenomic sequencing has recently become interesting [107,110]. Metagenomic sequencing does not target a single microorganism or bacterium, but a mixture of genomes from various microorganisms or bacteria. For example, it can be used to classify bacterial populations in clinical samples of patients without purification. In addition to such medical applications, the environmental impact of genetically modified organisms (GMOs) by the monitoring of the rate of genetic modification in plants is a potential important target. The monitoring of hazardous substances such as bacteria, viruses, and explosives are also important targets. There have been reports on the detection of harmful ions [44,47,48] and explosives such as TNT [112].

Discussion and Future Prospects
In order to increase applications of single-molecule measurement by nanodevice, further integration of functional nanostructures and improvements and the simplification of analysis methods are required. Some approaches to these issues are described in the following paragraphs.
The first approach is the integration of various functional nanostructures on nanodevice sensors. For instance, nanochannels have made great achievements in sample control and transport by electrophoresis in sensor devices [113][114][115] and nanopillar structure serve as purification, separation, and transportation [116,117]. Along with the integration of nanodevices, the development of fabrication techniques for integrated nanodevices is an important issue. In nanodevices, if the shape of the fabricated device can be parallelized with high accuracy, the throughput of measurement can be expected to be dramatically improved. So far, parallelization has been reported for nanopore and nanogap devices [118,119].
The second approach is to use nanochannel or nanowell structures for optical sensor detection. For example, a single molecule in a nanochannel can be detected by optical microscopy with high sensitivity [120][121][122][123]. There are also zero-mode waveguides using the near-field effect and Raman spectroscopy using the plasmon phenomenon in the nanogap [124][125][126][127][128]. These nano-optics/electronics hybrid devices would improve the sensing selectivity.
The third approach is the development of bioinformatics analysis methods using artificial intelligence (AI). In single-molecule measurements, the data volume is exponentially increased because of the high-speed data acquisition and large number of detected molecular signals in sample solutions, compared to conventional analytical methods. Moreover, the detected signal shape, i.e., electrical current-time profile, become more complex. Therefore, AI-based informatics analysis methods are suitable for sample identifications as the method enables the extraction of characteristic parameters from complex signals. In recent years, AI-based analysis has made it possible to identify target molecules among similar structural molecules [10,111,[129][130][131]. Thus, it can be said that the evolution of bioinformatics analysis is essential for single-molecule measurement by nanodevice.
In addition to these developments of nanodevice based technologies and methods, there are several steps that precede real clinical practice use. The first step is the standardization of measurement systems, the nanodevices, and the data-analysis methods. The next step is to obtain medical approval from the government in each country. In the current stage, nanodevice methods are currently taking these steps towards application in the medical field. For example, the Food and Drug Administration (FDA) has issued guidelines for obtaining medical approval for next-generation sequencing nanodevices such as MinION [132][133][134]. Furthermore, the recent COVID-19 pandemic has accelerated the movement to detect viruses (SARS-CoV-2) and virus mutants by nanodevices. It is expected that the application of this technology will expand as this trend becomes more active in the future.
In summary, the single-molecule electrical measurement by nanodevices is an interdisciplinary field that is currently undergoing development while incorporating new technologies. I believe that single-molecule measurement methods using nanodevices will greatly develop as a key technology for realizing personal medical care that enables pointof-care, which is considered a dream come true, while expanding the range of its applications.
Funding: This research was funded by Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (21H01741).

Institutional Review Board Statement:
Not applicable for studies not involving humans or animals.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.