Enhancement of electrode performance through surface modification using carbon nanotubes and porous gold nanostructures.

Recently, the demand for the sensitive detection of nanomaterials and biomolecules has been increasing for evaluating the toxicity of nanomaterials and early diagnosis of diseases. Although many studies have developed new detection assays, these are heavily influenced by the capabilities of the detection equipment. Therefore, the aim of the present study was to improve electrode performance by modifying the surface of the detection electrode using a simple method. Electrode surface modification was performed using carbon nanotubes (CNT) and porous gold nanostructures (NS) with excellent electrical and chemical properties. Through the simple physical deposition of CNT and electrochemical reduction of NS, the increasement of the electrode surface area was achieved. Because of the CNTs attached to the electrodes at the first step, the metal ions constituting the NS can adhere well to the electrodes. Nanoparticles with a porous structure can be generated through electrochemical reduction (cyclic voltammetry) of metal ions attached to electrodes. Consequently, the surface area of the electrode increased and electrochemical performance was improved (confirmed by atomic force microscopy, Nyquist plot and Bode plot). To quantitatively confirm the improvement of electrode performance according to the surface change through the proposed treatment technique, DNA was detected. Unlike previous surface modification studies, the developed surface treatment technique can be applied to a variety of detection equipment. To confirm this, the detection was performed using two detection devices with different operating principles. DNA detection using the two types of equipment confirmed that the detection limit was increased by approximately 1000-fold through applying a simple surface treatment. In addition, this method is applicable to detect various sizes of nanomaterials. The method proposed in this study is simple and has the advantage that it can be applied to various devices and various materials.

A microcantilever-based silver ion sensor using DNA-functionalized gold nanoparticles as a mass amplifier.

Silver ions have been used to sterilize many products, however, it has recently been demonstrated that silver ions can be toxic. This toxicity has been studied over many years with the lethal concentration at 10 µM. Silver ions can accumulate through the food chain, causing serious health problems in many species. Hence, there is a need for a commercially available silver ion sensor, with high detection sensitivity. In this work, we develop an ultra-sensitive silver ion sensor platform, using cytosine based DNA and gold nanoparticles as the mass amplifier. We achieve a lower detection limit for silver ions of 10 pM; this detection limit is one million times lower than the toxic concentration. Using our sensor platform we examine highly selective characteristics of other typical ions in water from natural sources. Furthermore, our sensor platform is able to detect silver ions in a real practical sample of commercially available drinking water. Our sensor platform, which we have termed a 'MAIS' (mass amplifier ion sensor), with a simple detection procedure, high sensitivity, selectivity and real practical applicability has shown potential as an early toxicity assessment of silver ions in the environment.

Highly sensitive, direct and real-time detection of silver nanowires by using a quartz crystal microbalance.

For several decades, silver nanomaterials (AgNMs) have been used in various research areas and commercial products. Among the many AgNMs, silver nanowires (AgNWs) are one of the mostly widely used nanomaterials due to their high electrical and thermal conductivity. However, recent studies have investigated the toxicity of AgNWs. For this reason, it is necessary to develop a successful detection method of AgNWs for protecting human health. In this study, label-free, highly sensitive, direct, and real-time detection of AgNWs is performed for the first time. The detection mechanism is based on the resonance frequency shift upon the mass change from the hybridization between the probe DNA on the electrode and the linker DNA attached on AgNWs. The frequency shift is measured by using a quartz crystal microbalance. We are able to detect 1 ng ml-1 of AgNWs in deionized water in real-time. Moreover, our detection method can selectively detect AgNWs among other types of one-dimensional nanomaterials and can also be applied to detection in drinking water.

Ultra-sensitive detection of zinc oxide nanowires using a quartz crystal microbalance and phosphoric acid DNA.

Recent advancements of nanomaterials have inspired numerous scientific and industrial applications. Zinc oxide nanowires (ZnO NWs) is one of the most important nanomaterials due to their extraordinary properties. However, studies performed over the past decade have reported toxicity of ZnO NWs. Therefore, there has been increasing demand for effective detection of ZnO NWs. In this study, we propose a method for the detection of ZnO NW using a quartz crystal microbalance (QCM) and DNA probes. The detection method is based on the covalent interaction between ZnO NWs and the phosphoric acid group of single-stranded DNA (i.e., linker DNA), and DNA hybridization between the linker DNA and the probe DNA strand on the QCM electrode. Rapid, high sensitivity, in situ detection of ZnO NWs was demonstrated for the first time. The limit of detection was 10(-4) µg ml(-1) in deionized water, which represents a sensitivity that is 100000 times higher than the toxic ZnO NW concentration level. Moreover, the selectivity of the ZnO NW detection method was demonstrated by comparison with other types of nanowires and the method was able to detect ZnO NWs in tap water sensitively even after stored for 14 d in a refrigerator. The performance of our proposed method was sufficient to achieve detection of ZnO NW in the 'real-world' environment.

A highly sensitive, direct and label-free technique for Hg(2+) detection using Kelvin probe force microscopy.

For several decades, various nanomaterials have been used in a wide range of industrial fields, research areas, and commercial products. Among many nanomaterials, nano-sized mercury materials are one of the most widely used nanomaterials in real life. However, due to the high toxicity of Hg(2+), it is imperative to develop an effective and practical detection method for Hg(2+) to protect human health and environment. In this study, a highly sensitive, label-free method of detecting Hg(2+) that requires only a single drop of solution was developed. The detection mechanism is based on the different surface potential arising from Hg(2+) binding to mismatched thymine-thymine sequences, creating a very stable base pair. The surface potential is measured with Kelvin probe force microscopy (KPFM) to a molecular resolution. The developed method is capable of detecting 2 fmol of Hg(2+), which is 500 times more sensitive than previously reported techniques. Moreover, our method can selectively detect Hg(2+) and can also be applied to tap water and river water. This KPFM-based Hg(2+) detection method can be used as an early detection technique for practical applications.

Enhanced selective discrimination of point-mutated viral RNA through false amplification regulatory direct insertion in rolling circle amplification.

Coronaviruses are single-stranded RNA viruses with high mutation rates. Although a diagnostic method for coronaviruses has been developed, variants appear rapidly. Low test accuracy owing to single-point mutations is one of the main factors in the failure to prevent the early spread of coronavirus infection. Although reverse transcription-quantitative polymerase chain reaction can detect coronavirus infection, it cannot exclude the possibility of false positives, and an additional multiplexing kit is needed to discriminate single nucleotide polymorphism (SNP) variants. Therefore, in this study, we introduced a new nucleic acid amplification method to determine whether an infected person has a SNP mutation using a lateral flow assay (LFA) as a point-of-care test. Unlike traditional DNA amplification methods, direct insertion into rolling circle amplification amplifies the target genes without false amplification. After SNP-selective nucleic acid amplification, nuclease enzymes are used to make double-stranded DNA fragments that the LFA can detect, where specific mismatched DNA is found and cleaved to show different signals when a SNP-type is present. Therefore, wild- and SNP-type variants can be selectively detected. In this study, the limit of detection was 400 aM for viral RNA, and we successfully identified a dominant SNP variant selectively. Clinical tests were also conducted.

Sensitive and selective DNA detecting electrochemical sensor via double cleaving CRISPR Cas12a and dual polymerization on hyperbranched rolling circle amplification.

Electrochemical sensors are widely used for nucleic acid detection. However, they exhibit low sensitivity and specificity. To overcome these limitations, DNA amplification method is necessary. In this study, we introduced CRISPR (Clustered regularly interspaced short palindromic repeats) Cas12a-dependent hyperbranched rolling circle amplification (HRCA) into an electrochemical sensor platform. By resolving the existing false-positive issue of HRCA, CRISPR Cas12a determines the real positive amplification that able to enhance its sensitivity for extremely low concentrations of nucleic acids and specificity for single-point mutations. In detail, CRISPR Cas12a, which activates the nucleic acid amplification reaction, was used for both trans and cis cleavage for the first time. Finally, selectively amplified DNA was detected using a screen-printed electrode. Using the change in surface coverage by DNA, the electrochemical sensor detected a decrease in the redox signal. In summary, combining a novel DNA amplification method and electrochemical sensor platform, our proposed method compensates for the shortcomings of existing RCA and hyperbranched RCA, secures a high sensitivity of 10 aM, and overcomes false-positivity problems. Moreover, such creative applications of CRISPR Cas12a may lead to the expansion of its applications to other nucleic acid amplification methods.

The work function of doped polyaniline nanoparticles observed by Kelvin probe force microscopy.

The work function of polyaniline nanoparticles in the emeraldine base state was determined by Kelvin probe force microscopy to be ~270 meV higher than that of similar nanoparticles in the emeraldine salt state. Normal tapping mode atomic force microscopy could not be used to distinguish between the particles due to their similar morphologies and sizes. Moreover, other potential measurement systems, such as using zeta potentials, were not suitable for the measurement of surface charges of doped nanoparticles due to their encapsulation by interfering chemical groups. Kelvin probe force microscopy can be used to overcome these limitations and unambiguously distinguish between the bare and doped polyaniline nanoparticles.

Novel Detection Method for Circulating EGFR Tumor DNA Using Gravitationally Condensed Gold Nanoparticles and Catalytic Walker DNA.

The detection of circulating tumor DNA is a major challenge in liquid biopsies for cancer. Conventionally, quantitative polymerase chain reactions or next-generation sequencing are used to detect circulating tumor DNA; however, these techniques require significant expertise, and are expensive. Owing to the increasing demand for a simple diagnostic method and constant monitoring of cancer, a cost-effective detection technique that can be conducted by non-experts is required. The aim of this study was to detect the circulating tumor DNA containing the epidermal growth factor receptor (EGFR) exon 19 deletion, which frequently occurs in lung cancer. By applying walker DNA to a catalytic hairpin assembly and using the differential dispersibility of gold nanoparticles, we detected EGFR exon 19 deletion mutant #2 DNA associated with lung cancer. Our sensing platform exhibited a limit of detection of 38.5 aM and a selectivity of 0.1% for EGFR exon 19 wild-type DNA. Moreover, we tested and compared EGFR exon 19 deletion mutants #1 and #3 to evaluate the effect of base pair mismatches on the performance of the said technique.

Highly Sensitive and Real-Time Detection of Zinc Oxide Nanoparticles Using Quartz Crystal Microbalance via DNA Induced Conjugation.

With the development of nanotechnology, nanomaterials have been widely used in the development of commercial products. In particular, zinc oxide nanoparticles (ZnONPs) have been of great interest due to their extraordinary properties, such as semiconductive, piezoelectric, and absorbance properties in UVA and UVB (280-400 nm) spectra. However, recent studies have investigated the toxicity of these ZnONPs; therefore, a ZnONP screening tool is required for human health and environmental problems. In this study, we propose a detection method for ZnONPs using quartz crystal microbalance (QCM) and DNA. The detection method was based on the resonance frequency shift of the QCM. In detail, two different complementary DNA strands were used to conjugate ZnONPs, which were subjected to mass amplification. One of these DNA strands was designed to hybridize to a probe DNA immobilized on the QCM electrode. By introducing the ZnONP conjugation, we were able to detect ZnONPs with a detection limit of 100 ng/mL in both distilled water and a real sample of drinking water, which is 3 orders less than the reported critical harmful concentration of ZnONPs. A phosphate terminal group, which selectively interacts with a zinc oxide compound, was also attached at one end of a DNA linker and was attributed to the selective detection of ZnONPs. As a result, better selective detection of ZnONPs was achieved compared to gold and silicon nanoparticles. This work demonstrated the potential of our proposed method as a ZnONP screening tool in real environmental water systems.

Synergistic enhanced rolling circle amplification based on mutS and radical polymerization for single-point mutation DNA detection.

The detection of nucleic acids in biofluids is essential for changing the paradigm of disease diagnosis. As there are very few nucleic acids present in human biofluids, a high sensitivity method is required to detect nucleic acids for disease diagnosis. The Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation is associated with non-small cell lung cancer. It is a point mutation and requires a highly selective detection technique. In this study, high sensitivity and selectivity were achieved for the detection of KRAS mutation using rolling circle amplification (RCA), atomic transfer radical polymerization (ATRP), mutS enzyme, and electrochemical sensors. Although RCA can isothermally amplify DNA, it has low selectivity for detecting single-base mismatch DNA, and its sensitivity is not suitable for circulating tumor DNA detection. The selectivity of RCA was improved by using mutS, which can bind specifically to point mutations. In addition, as a method of isothermal radical polymerization, ATRP was used to amplify the weak signal of RCA. Since RCA and ATRP reactions occur simultaneously, detection time was reduced, and the calculated detection limit was 3.09 aM. Computational and experimental analyses were conducted to verify each detection step and the combination of mutS, ATRP, and RCA. The experiment was performed using normal human serum samples for biological application, and the proposed detection method was confirmed to have excellent potential for diagnosing cancer patients.

Ultrahigh sensitive and selective detection of single nucleotide polymorphism using peptide nucleic acid and ribonuclease H assembled DNA amplification (PRADA).

Early diagnosis and monitoring of cancer is the best way to increase the survival rate among patients with cancer. However, the current cancer screening technology is expensive and time-consuming; hence, cancer screening remains challenging. Therefore, developing a relatively inexpensive and high-performance analytical method is necessary. Especially, mutations in KRAS can be characterized as single nucleotide polymorphism mutations. Therefore, discrimination of single nucleotide polymorphism is essential to detect cancer mutations. This study introduces a method with high sensitivity and selectivity of real-time PCR using peptide nucleic acid (PNA) and RNase H II to detect KRAS single nucleotide polymorphism. This method was developed via the fusion of the existing PNA clamping PCR technique and the RNase H-dependent PCR technique. A synergistic effect was realized by mitigating the shortcomings of each method. Our method had a detection limit of 1 aM and selectivity of 0.01%. This study demonstrated completed validation tests with DNA-spiked plasma sample analysis, cell culture, and analysis of blood samples collected from patients with cancer. Furthermore, we demonstrated the applicability of this method for breath biopsy.

Highly sensitive and selective detection of single-nucleotide polymorphisms using gold nanoparticle MutS enzymes and a micro cantilever resonator.

Mutant DNAs are important markers useful for the diagnosis of human disease. Single-nucleotide polymorphisms (SNPs) represent the most common types of DNA mutations. As there is only a one base pair change in a single nucleotide between the SNP and the wild-type DNA, it is difficult to distinguish the SNPs. In this report, a highly sensitive and selective detection and discrimination of SNPs is performed using MutS, gold nanoparticles (AuNP) and a resonator. A single mismatched base exists between the SNP mutation and the probe DNA on the resonator, and MutS binds to the DNA at the location of the mismatch. As MutS is attached to AuNP (MutS-AuNP), both MutS and AuNP are adsorbed onto the resonator. The detection is based on the resonance frequency shift of the resonator following the adsorption of MutS-AuNP on the resonator. Highly sensitive detection of DNA mutations was achieved using AuNPs that act as mass amplifiers, and the obtained limit of detected was 100 fM. Additionally, our proposed method detected mutations in the presence of as little as 0.1% wild-type, and discrimination of specific mutations was also achieved. The results obtained from our proposed method suggest its potential for diagnosing cancer patients.

Label-free and high-sensitive detection of Kirsten rat sarcoma viral oncogene homolog and epidermal growth factor receptor mutation using Kelvin probe force microscopy.

Assessment of Kirsten rat sarcoma viral oncogene homolog (KRAS) and epidermal growth factor receptor (EGFR) mutations are essential for targeted therapies of patients with non-small-cell lung cancer. In this report, we propose a label-free and high-sensitive detection method of KRAS and EGFR mutations using KPFM and a gold nanoparticle (AuNP)-based platform that densely adsorbs probe DNA and minimizes the sensing area. The detection is based on the evaluation of the surface potential of each AuNP. When AuNPs are modified with probe DNA (AuNP-pDNA), the surface potential is shifted towards the negative potential due to the negatively charged DNA backbone. When AuNP-pDNA further captures target mutant DNA through DNA hybridization, an additional surface potential shift occurs. The platform is able to detect KRAS mutant DNA (13 mer) and EGFR mutant DNA (84 mer) with a limit of detection (LOD) of 3.3pM. Furthermore, the platform is able to detect selectively the KRAS mutant DNA from its wild-type DNA. Our proposed label-free and high-sensitive KPFM method has shown potential glimpses of a personalized medical diagnosis for cancer patients.

Label-free detection of zinc oxide nanowire using a graphene wrapping method.

Zinc oxide nanowires (ZnO NWs) have been attempted to various applications, such as piezoelectric devices, energy harvesting devices, self-powered nanosensors, and biomedical devices. However, recent reports have shown the toxic effect of ZnO NWs. In this report, we described the detection of ZnO NWs, for the first time using reduced graphene oxide (RGO) wrapping method. By wrapping RGO to ZnO NW (RGO-ZnO NW), we are able to aggregate ZnO NWs and increase the sensing performance. The detection measurement is based on the resonance frequency shift derived from mass variation of RGO-ZnO NW adsorption on the DNA immobilized resonator. The resonator is able to detect ZnO NWs with detection limit of 100 ng mL(-1) which is 2 order below the fatal toxic concentration of ZnO NWs in Human Monocyte Macrophages (HMMs). Furthermore, the resonator is able to detect ZnO NWs in real tap water, showing the potential as ZnO NWs screening platform in real environmental aqua system.

Ultra-sensitive direct detection of silver ions via Kelvin probe force microscopy.

Nanotoxicity is receiving great importance due to its potential impact on human health and environment and due to rapid development in the field of nanoscale research and industry. Herein, we report the Kelvin probe force microscope (KPPM)-based nanotoxicity material detection using surface potential difference. In general, it is difficult to measure the size of ion (Ag(+)) using a conventional atomic force microscope (AFM) because of the limited resolution. In this study, we have demonstrated that KPFM is capable of ultra-sensitive detection of silver ion with silver specific DNA by a single droplet. Furthermore, the measured surface potentials for Ag+ and DNA binding enable the detection performance for a practical sample that is general drinking water. Remarkably, the KPFM based silver ion detection enables an insight into the coordination chemistry, which plays an important role in early detection of toxicity. This implies that KPFM based detection system opens a new avenue for water testing sensor.

Multimodal label-free detection and discrimination for small molecules using a nanoporous resonator.

To detect chemical or biological threats, it is crucial that sensor devices can differentiate various target molecules. In general, each different sensing method has its own strengths and weaknesses due to their respective limitations. For example, although resonant sensors have high sensitivity, they are not able to discriminate target molecules. At the same time, although surface-enhanced Raman spectroscopy is a representative label-free detection method that can discriminate target molecules, its fabrication is often complex and expensive. Here we present a label-free multimodal nanoporous resonator-based system for small molecule detection and discrimination that combines the strengths of each of these sensing methods. Our approach is not only able to improve the sensitivity of the resonant sensor but it can also discriminate the target molecules. Furthermore, the fabrication process is swift (lasting <3 min) and convenient.

High-sensitivity detection of silver ions using oligonucleotide-immobilized oscillator.

With the remarkable developments in the fields of nanoscale research and industry, nanotoxicity is gaining importance from the viewpoint of its potential impact on human health and the environment. Herein, we report on the label-free, high-sensitivity detection of Ag(+), a representative nanotoxic material, by using a silver-specific nucleotide-coated oscillator. The detection is based on the measurement of the resonant frequency shift arising from constitution of the cytosine-Ag(+)-cytosine bonding. We amplify the resonant frequency shift by using single cytosine molecules. It is shown that a silver-specific DNA-immobilized oscillator enables the capture of silver ions at concentrations below 1 nM. Remarkably, the nucleotide-based oscillator enables an insight into the coordination chemistry, which plays an important role in the early detection of toxicity. This implies that the bio-conjugated sensor could be used to set the reference point for water quality.

Highly sensitive detection of self-aggregated single-walled carbon nanotubes using a DNA-immobilized resonator.

By using DNA linkers, we are able to conjugate self-crosslinked SWNTs which could be detected upon hybridization with a DNA immobilized resonator. The DNA immobilized resonator is able to detect SWNTs with a detection limit of 10 ng ml(-1) which was 2-3 orders of magnitude smaller than the reported SWNT toxicity concentration.