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Tomohiro Murata, [Kosuke Minami](https://orcid.org/0000-0003-4145-1118), [Tomohiko Yamazaki](https://orcid.org/0000-0003-2136-8042), [Genki Yoshikawa](https://orcid.org/0000-0002-9136-8964), [Katsuhiko Ariga](https://orcid.org/0000-0002-2445-2955)

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[Detection of Trace Amounts of Water in Organic Solvents by DNA-Based Nanomechanical Sensors](https://mdr.nims.go.jp/datasets/27d0e5e7-7926-402e-ade4-4c784a83cb15)

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Detection of Trace Amounts of Water in Organic Solvents by DNA-Based Nanomechanical SensorsCitation: Murata, T.; Minami, K.;Yamazaki, T.; Yoshikawa, G.; Ariga, K.Detection of Trace Amounts of Waterin Organic Solvents by DNA-BasedNanomechanical Sensors. Biosensors2022, 12, 1103. https://doi.org/10.3390/bios12121103Received: 2 November 2022Accepted: 28 November 2022Published: 1 December 2022Publisher’s Note: MDPI stays neutralwith regard to jurisdictional claims inpublished maps and institutional affil-iations.Copyright: © 2022 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).biosensorsArticleDetection of Trace Amounts of Water in Organic Solvents byDNA-Based Nanomechanical SensorsTomohiro Murata 1,2, Kosuke Minami 3,* , Tomohiko Yamazaki 4,5 , Genki Yoshikawa 3,6and Katsuhiko Ariga 1,2,*1 Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa 277-8561, Japan2 International Center for Materials Nanoarchitectonics (MANA), National Institute for MaterialsScience (NIMS), 1-1 Namiki, Tsukuba 305-0044, Japan3 Center for Functional Sensor & Actuator (CFSN), Research Center for Functional Materials (RCFM),National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba 305-0044, Japan4 Research Center for Functional Materials (RCFM), National Institute for Materials Science (NIMS),1-2-1 Sengen, Tsukuba 305-0047, Japan5 Division of Life Science, Hokkaido University, Kita 10, Nishi 8, Kita-ku, Sapporo 060-0808, Japan6 Materials Science and Engineering, Graduate School of Pure and Applied Science, University of Tsukuba,1-1-1 Tennodai, Tsukuba 305-8571, Japan* Correspondence: minami.kosuke@nims.go.jp (K.M.); ariga.katsuhiko@nims.go.jp (K.A.)Abstract: The detection of trace amounts of water in organic solvents is of great importance in thefield of chemistry and in the industry. Karl Fischer titration is known as a classic method and iswidely used for detecting trace amounts of water; however, it has some limitations in terms ofrapid and direct detection because of its time-consuming sample preparation and specific equipmentrequirements. Here, we found that a DNA-based nanomechanical sensor exhibits high sensitivityand selectivity to water vapor, leading to the detection and quantification of trace amounts of waterin organic solvents as low as 12 ppm in THF, with a ppb level of LoD through their vapors. Sincethe present method is simple and rapid, it can be an alternative technique to the conventional KarlFischer titration.Keywords: DNA; nanomechanical sensors; membrane-type surface stress sensor (MSS); water detection;trace amounts of water in organic solvent1. IntroductionDNA is one of the most interesting biopolymers in science. Besides its central functionas the storage and carrier of genetic information, its unique nature offers great potential inmany fields. One of the most important properties of DNA is its hybridization capability,such as the ability to form double helices and guanine quadruplexes. DNA nanotech-nology has been extensively studied [1–3], and versatile potential applications includingtherapeutics [4,5] and diagnosis [6–8] have been reported. In particular, DNA hybridiza-tion capability has been exploited in the field of biosensors to detect nucleic acids withhigh selectivity [9–12]. Another important property of DNA is its hydration behavior [13].The hydration/dehydration behavior is known to affect the hybridization process andthe derived structures [14–17] as well as the material properties, such as the mechanicalcharacteristics [15,18,19].Focusing on the changes in mechanical properties associated with DNA hydration,nanomechanical sensors have attracted attention as potential sensing platforms to detectthe mechanical deformation of receptor layers caused by the sorption of target moleculesand hence have been applied to measuring DNA hydration [17,18,20], mechanical prop-erties [18,19] and hybridization [18,21]. The hybridization behavior of DNA has led tothe development of DNA-based nanomechanical sensors for various applications [22–26].Biosensors 2022, 12, 1103. https://doi.org/10.3390/bios12121103 https://www.mdpi.com/journal/biosensorshttps://doi.org/10.3390/bios12121103https://doi.org/10.3390/bios12121103https://creativecommons.org/https://creativecommons.org/licenses/by/4.0/https://creativecommons.org/licenses/by/4.0/https://www.mdpi.com/journal/biosensorshttps://www.mdpi.comhttps://orcid.org/0000-0003-4145-1118https://orcid.org/0000-0003-2136-8042https://orcid.org/0000-0002-9136-8964https://orcid.org/0000-0002-2445-2955https://doi.org/10.3390/bios12121103https://www.mdpi.com/journal/biosensorshttps://www.mdpi.com/article/10.3390/bios12121103?type=check_update&version=1Biosensors 2022, 12, 1103 2 of 11Although there is a limited number of papers using DNA as a receptor material for nanome-chanical sensors to detect water vapor, albeit for different purposes (i.e., the detectionof hydration) [17,20], DNA-based nanomechanical sensors have mostly been applied inaqueous environment because of their potential applications [24,25].Moreover, in hydration, a limited number of water molecules hydrate the phosphategroups in the DNA backbone [13]. Since this hydration can be detected by nanomechanicalsensors [17,18,20], it has great potential as a sensor to detect trace amounts of water,especially in organic solvents, which is of great importance in the field of chemistry andin the industry. Although there are several methods to detect trace amounts of water inorganic solvents, such as conventional coulometric and volumetric analyses, known as KarlFischer titration [27–29], as well as colorimetric approaches [30,31] and others [32,33], tothe best of our knowledge, there have been few reports on detecting trace amounts of waterin organic solvents through their vapors.In this work, we found that a DNA-based nanomechanical sensor exhibited high selec-tivity to water vapor. We used one of the nanomechanical sensors operating in the so-calledstatic mode, a Membrane-type Surface stress Sensor (MSS) [26,34], and deposited naturalDNA obtained from salmon testes onto the MSS. Compared to nanomechanical sensors indynamic mode operation [25] as well as to other types of gas and chemical sensors [35],nanomechanical sensors in static mode operation detect mechanical stress/strain [26] andhence transduce the mechanical deformation induced by the swelling of a DNA layerassociated with DNA hydration, resulting in high sensitivity to water. Owing to the highsensitivity and selectivity of the examined sensor to water vapor, we also demonstrate inthis work that trace amounts of water in organic solvents can be detected. This study notonly presents the possibility of using DNA as a water-sensitive receptor material but alsoprovides a promising sensing platform for detecting trace amounts of water in organicsolvents as an alternative to the conventional Karl Fischer titration method [27–29].2. Materials and Methods2.1. MaterialsDNA from salmon testes was purchased from Tokyo Chemical Industry CO., LTD.Ultrapure water for inkjet spotting and sensing measurements was prepared by MilliporeMilli-Q. Acetone, methanol, ethanol, acetonitrile, n-hexane, benzene, toluene, tetrahydro-furan (THF), and pyridine for sensing measurements were purchased from FUJIFILMWako Pure Chemical Corporation. All solvents were dehydrated (H2O < 0.001–0.005%).Molecular sieves 3A were purchased from FUJIFILM Wako Pure Chemical Corporation.Unless otherwise noted, the materials were used as purchased.2.2. Fabrication of DNA-Coated MSSThe construction of the MSS chips and their working principle have been previouslyreported [34,36]. Briefly, the MSS consists of a silicon-based membrane suspended by foursensing beams, composing a full Wheatstone bridge (Figure 1A). In each sensing beam,piezoresistors were embedded by boron doping. The membrane is coated with a receptormaterial (in the present study, DNA). When the receptor layer deforms upon the sorptionof the target analytes, the receptor layer generates surface stress on the membrane [26]. Thesurface stress is transduced to the four sensing beams as amplified uniaxial stress, resultingin changes in the electrical resistance of the piezoresistors embedded in the beams. Thesignal output of the MSS (Vout) is provided by the total output resistance change obtainedfrom the Wheatstone bridge circuit; it can be expressed asVout =VB4(∆R1R1− ∆R2R2+∆R3R3− ∆R4R4)(1)where VB is the bridge voltage applied on the Wheatstone bridge circuit, and ∆R/Ri(i = 1–4) is the relative resistance change in each sensing beam.Biosensors 2022, 12, 1103 3 of 11Biosensors 2022, 12, x FOR PEER REVIEW 3 of 11  31 2 4out1 2 3 44BRV R R RVR R R R   = + −− , (1) where VB is the bridge voltage applied on the Wheatstone bridge circuit, and ΔR/Ri (i = 1–4) is the relative resistance change in each sensing beam.  The MSS chips used in the present study were purchased from NanoWorld AG, Swit-zerland. The dimensions of the MSS used in this study are shown in Figure 1A. Before the deposition of DNA, the MSS chips were treated with oxygen plasma using a low-pressure plasma system (Femto, version B, Diener Electronic GmbH + Co. KG., Ebhausen, Ger-many). The plasma power, pressure, and duration were 30 W, 0.50 mbar, and 2 min, re-spectively. DNA was deposited directly on the membrane of the MSS by using an inkjet spotter (LaboJet-500SP, MICROJET Corporation) with a nozzle (IJHBS-300, MICROJET Corporation). DNA was dissolved in ultrapure water at a concentration of 0.2 mg mL–1, and the resulting solution was deposited onto each channel of the MSS. The discharged volume per shot by the inkjet was 492 ± 15 pL (n = 3) at this concentration.  Figure 1. Schematic illustrations of the MSS and its sensing system. (A) Configuration of the MSS. Sensing beams with embedded piezoresistors are magnified in the insets. Numbers indicate the di-mensions in µm. (B) Schematic illustration of the measurement system. 2.3. Characterization of the DNA Film The thickness and surface profile of the DNA films coated on the MSS were measured by using a 3D surface profiler (VK-X3000, KEYENCE Corporation, Osaka, Japan) under the laser confocal mode and a surface stylus profiler (DekTak, Bruker). To confirm the structure of DNA, the circular dichroism (CD) spectra of an aqueous solution and a drop-casted film of DNA were measured using a spectropolarimeter (J-820, JASCO). An aque-ous solution of DNA (5 mg mL–1) was drop-casted on a quartz substrate. The base-pair lengths of the DNA molecules were confirmed by electrophoresis. Electrophoresis was performed using a polyacrylamide gel (e-PAGEL 10–20%, ATTO) in TG buffer at 21 mA for 65 min (WSE-1150 PageRunAce, ATTO). Ultra-Low-Range DNA Ladder (Thermo Fisher Scientific) was used for the quantification based on base size. The DNA samples were stained for 30 min in TBE buffer with SYBR™ Gold (Thermo Fischer Scientific, Wal-tham, MA, USA). 2.4. Preparation of Dehydrated Organic Solvents and Water-Contaminated Solvents Dehydrated organic solvents were stored with molecular sieves 3A under nitrogen during the experiment; the molecular sieves were activated before use by heating in an oven at 250 °C overnight and then cooling to room temperature under vacuum for 3 h. To prepare the water-contaminated solvents, an aliquot of water was added to the dehy-drated organic solvents. To obtain a series of low concentrations, the resulting water-con-taminated solvents were further diluted with the dehydrated organic solvents prepared above. Figure 1. Schematic illustrations of the MSS and its sensing system. (A) Configuration of the MSS.Sensing beams with embedded piezoresistors are magnified in the insets. Numbers indicate thedimensions in µm. (B) Schematic illustration of the measurement system.The MSS chips used in the present study were purchased from NanoWorld AG,Switzerland. The dimensions of the MSS used in this study are shown in Figure 1A. Beforethe deposition of DNA, the MSS chips were treated with oxygen plasma using a low-pressure plasma system (Femto, version B, Diener Electronic GmbH + Co. KG., Ebhausen,Germany). The plasma power, pressure, and duration were 30 W, 0.50 mbar, and 2 min,respectively. DNA was deposited directly on the membrane of the MSS by using an inkjetspotter (LaboJet-500SP, MICROJET Corporation, Nagano, Japan) with a nozzle (IJHBS-300,MICROJET Corporation). DNA was dissolved in ultrapure water at a concentration of0.2 mg mL−1, and the resulting solution was deposited onto each channel of the MSS. Thedischarged volume per shot by the inkjet was 492 ± 15 pL (n = 3) at this concentration.2.3. Characterization of the DNA FilmThe thickness and surface profile of the DNA films coated on the MSS were measuredby using a 3D surface profiler (VK-X3000, KEYENCE Corporation, Osaka, Japan) under thelaser confocal mode and a surface stylus profiler (DekTak, Bruker). To confirm the structureof DNA, the circular dichroism (CD) spectra of an aqueous solution and a drop-casted filmof DNA were measured using a spectropolarimeter (J-820, JASCO). An aqueous solutionof DNA (5 mg mL−1) was drop-casted on a quartz substrate. The base-pair lengths of theDNA molecules were confirmed by electrophoresis. Electrophoresis was performed using apolyacrylamide gel (e-PAGEL 10–20%, ATTO) in TG buffer at 21 mA for 65 min (WSE-1150PageRunAce, ATTO). Ultra-Low-Range DNA Ladder (Thermo Fisher Scientific) was usedfor the quantification based on base size. The DNA samples were stained for 30 min in TBEbuffer with SYBR™ Gold (Thermo Fischer Scientific, Waltham, MA, USA).2.4. Preparation of Dehydrated Organic Solvents and Water-Contaminated SolventsDehydrated organic solvents were stored with molecular sieves 3A under nitrogenduring the experiment; the molecular sieves were activated before use by heating in anoven at 250 ◦C overnight and then cooling to room temperature under vacuum for 3 h. Toprepare the water-contaminated solvents, an aliquot of water was added to the dehydratedorganic solvents. To obtain a series of low concentrations, the resulting water-contaminatedsolvents were further diluted with the dehydrated organic solvents prepared above.2.5. Vapor SensingThe vapor sensing system is shown in Figure 1B. The DNA-coated MSS chips wereplaced in a Teflon-based chamber. The chamber was connected to two gas lines: an inletand an outlet. The inlet was connected to a gas system, which consisted of two mass flowcontrollers (MFCs), a mixing chamber, a purging gas line, and a sampling gas line with avial for a solvent liquid. The gas flow system with the chamber was placed in an incubator,and the temperature was maintained at 25.0 ± 0.5 ◦C. The headspace vapor of each solventBiosensors 2022, 12, 1103 4 of 11was introduced by a carrier gas. Dry and pure nitrogen gas was used as a carrier andpurging gas. The duration was precisely controlled using the two MFCs. Before measuringthe MSS output, pure nitrogen gas was introduced into the MSS chamber for at least 5 min(i.e., MFC-2 at 100 mL min−1) to promote the desorption of molecules absorbed in the previousmeasurement. Subsequently, MFC-1 (injection line) was controlled at 80 mL min−1 for 5 minand then switched off (i.e., 0 mL min−1) for 5 min. The total flow rate was adjusted to100 mL min−1 by controlling MFC-2 during the sensing experiments. The measurementsequence is shown in Figure S1. The data were obtained with the applied bridge voltageVB of –0.5 V and recorded at a rate of 20 Hz.To investigate the selectivity of DNA to various vapors, each vapor was introducedat the concentration Pa/Po of 0.8, where Pa and Po denote the partial vapor pressure andsaturated vapor pressure of each solvent, respectively. For the measurement of traceamounts of water in organic solvents, each vapor of organic solvents with 0–4000 ppmwater added by weight was injected to the chamber.2.6. Measurement of the Humidity DependenceThe signal changes of the DNA-deposited MSS at several humidity values wereexamined. Water was set as the solvent (Figure 1), and the value of MFC-1 was changedin the range of 0–90% RH. The total flow rate was adjusted to 100 mL min−1 by MFC-2.To estimate the strain of the DNA membrane due to water adsorption, a quartz crystalmicrobalance (QCM; QCM922A, SEIKO EG&G) measurement of the several humidityconditions was also conducted. An AT-cut quartz crystal resonator (QA-A9M-AU(M),SEIKO EG&G) was used and held into a QCM chamber (QA-CL6, SEIKO EG&G) insteadof the MSS chamber, as shown in Figure 1. The same amount of DNA solution used for theMSS was deposited on the resonator by the inkjet method mentioned before.2.7. Numerical SimulationThe signal responses of nanomechanical sensors operating in static mode includingMSS are obtained by measuring the mechanical stress/strain induced by the sorption oftarget molecules in a receptor layer (in this study, DNA). Generally, the volume of absorbedmolecules is one of the important factors to generate mechanical strain in a receptor layer.To simulate the effects of the volume-dependent strain of absorbed water on the MSSresponses, we performed finite element analysis (FEA) using COMSOL Multiphysics 5.6with the Structural Mechanics module according to our previous studies [37,38]. Thedimensions of the MSS are shown in Figure 1A. The volume of absorbed water moleculeswas estimated through the QCM measurement. Each structure of the DNA-coated MSSwas meshed with 10,000–20,000 elements to ensure numerical accuracy for identifyingmechanical deformation upon the applied strain and corresponding resistance changes.3. Results and Discussion3.1. Fabrication of the DNA-Coated MSSSince nanomechanical sensors including the MSS can obtain efficient signal responsesto sorption-induced mechanical deformation of a bulk receptor material [26,39], we coatedDNA on the membrane of an MSS by an inkjet spotter. The coating thickness was variedby changing the number of droplets of inkjet spotting (N) in the range from 100 to 1000.Optical laser microscope images are shown in Figure 2A. From the height profiles of theDNA films, the thicknesses of the DNA films were estimated to be approximately 100 nmto 10 µm (Figure 2B,C and Figure S2 in the Supplementary Materials). Although the DNAfilms on the MSS formed a coffee ring structure, as can be seen in Figure 2B, an MSS issufficiently robust with respect to the coating quality and inhomogeneity of a receptor layerowing to its symmetric geometry, and the signal deviation is in the range of only 5–6% evenwith a receptor layer having a coffee ring structure [40]. We used these DNA-coated MSS forfurther experiments. To verify the thickness-dependent sensitivity of the DNA-coated MSS,we measured the signal responses to water vapor. As shown in Figure 2D, the intensityBiosensors 2022, 12, 1103 5 of 11monotonically increased with a linear correlation up to N = 500, whereas the intensity forN = 1000 largely deviated, and the intensity at 20% RH was lower than that for N = 500.Thus, we used a DNA-coated MSS with N = 500 in the following experiments.Biosensors 2022, 12, x FOR PEER REVIEW 5 of 11  100 to 1000. Optical laser microscope images are shown in Figure 2A. From the height profiles of the DNA films, the thicknesses of the DNA films were estimated to be approx-imately 100 nm to 10 µm (Figure 2B,C and Figure S2 in the Supplementary Materials). Although the DNA films on the MSS formed a coffee ring structure, as can be seen in Figure 2B, an MSS is sufficiently robust with respect to the coating quality and inhomo-geneity of a receptor layer owing to its symmetric geometry, and the signal deviation is in the range of only 5–6% even with a receptor layer having a coffee ring structure [40]. We used these DNA-coated MSS for further experiments. To verify the thickness-dependent sensitivity of the DNA-coated MSS, we measured the signal responses to water vapor. As shown in Figure 2D, the intensity monotonically increased with a linear correlation up to N = 500, whereas the intensity for N = 1000 largely deviated, and the intensity at 20% RH was lower than that for N = 500. Thus, we used a DNA-coated MSS with N = 500 in the following experiments. We also characterized the DNA used in this work. Since the natural DNA from salmon testes used in this work was used as purchased, the sample contained DNA frag-ments of various lengths, as shown in Figure S3. According to the CD spectra, the DNA cast on quartz maintained its double-helix structure (Figure 2E,F). During inkjet spotting and the sensing measurements, aqueous solutions of DNA and the DNA-coated MSS were not subjected to any heating processes, suggesting that the DNA maintained its double-helix structure on the MSS.  Figure 2. DNA-coated MSS. (A) Optical microscope images of the DNA-coated MSS with a different number of inkjet droplets (N) ranging from 100 to 1000. Scale bar is 100 µm. (B) Height profile of the DNA film on MSS. N = 500. See also Figure S2 for all height profiles. (C) Plot of maximum thick-ness of DNA coated on the MSS as a function of a number of inkjet droplets (N). (D) Sensitivity to water as a function of DNA film thickness. The relative humidity varied as follows: 20%RH (green), 40%RH (blue), 60%RH (red), and 80%RH (black). (E,F) UV–Vis (E) and CD spectra (F) of a DNA aqueous solution (60 μg/mL) (blue) and DNA cast on quartz (red). CD spectra of DNA cast on quartz, magnified 10 times. Figure 2. DNA-coated MSS. (A) Optical microscope images of the DNA-coated MSS with a differentnumber of inkjet droplets (N) ranging from 100 to 1000. Scale bar is 100 µm. (B) Height profile ofthe DNA film on MSS. N = 500. See also Figure S2 for all height profiles. (C) Plot of maximumthickness of DNA coated on the MSS as a function of a number of inkjet droplets (N). (D) Sensitivityto water as a function of DNA film thickness. The relative humidity varied as follows: 20%RH(green), 40%RH (blue), 60%RH (red), and 80%RH (black). (E,F) UV–Vis (E) and CD spectra (F) of aDNA aqueous solution (60 µg/mL) (blue) and DNA cast on quartz (red). CD spectra of DNA cast onquartz, magnified 10 times.We also characterized the DNA used in this work. Since the natural DNA from salmontestes used in this work was used as purchased, the sample contained DNA fragmentsof various lengths, as shown in Figure S3. According to the CD spectra, the DNA cast onquartz maintained its double-helix structure (Figure 2E,F). During inkjet spotting and thesensing measurements, aqueous solutions of DNA and the DNA-coated MSS were notsubjected to any heating processes, suggesting that the DNA maintained its double-helixstructure on the MSS.3.2. Selectivity of the DNA-Coated MSSTo estimate the selectivity of the DNA-coated MSS to water, we measured 10 differentvapors, including 6 water-miscible organic solvents (i.e., methanol, ethanol, THF, acetone,acetonitrile, and pyridine), 3 water-immiscible solvents (n-hexane, benzene, and toluene),and water. The signal responses of the DNA-coated MSS to each vapor are shown inFigure 3A (see also Figure S4), and the corresponding signal outputs at 5 min after injection(denoted as signal intensity) are summarized in Figure 3B. As clearly seen in Figure 3A,B,the DNA-coated MSS exhibited a significantly high sensitivity to water with ∆R/R~0.35(Vout~44 mV; see also Equation (1)), while the sensitivity to other organic solvents was ca.Biosensors 2022, 12, 1103 6 of 1115–20 times lower, despite their hydrophilicity as well as water-miscibility. Interestingly,the response to benzene, which has a planer structure and is known as a DNA interca-lator, exhibited a similar intensity to those measured for other organic solvent vapors.According to the working principle of nanomechanical sensors in static mode operation,the signal output is obtained by the surface stress induced by mechanical deformation [26].Thus, the intercalation of benzene between the base pairs of DNA seemed to have lesseffect on mechanical deformation (i.e., expansion of DNA structure), resulting in a lowsignal response.Biosensors 2022, 12, x FOR PEER REVIEW 6 of 11  3.2. Selectivity of the DNA-Coated MSS To estimate the selectivity of the DNA-coated MSS to water, we measured 10 differ-ent vapors, including 6 water-miscible organic solvents (i.e., methanol, ethanol, THF, ac-etone, acetonitrile, and pyridine), 3 water-immiscible solvents (n-hexane, benzene, and toluene), and water. The signal responses of the DNA-coated MSS to each vapor are shown in Figure 3A (see also Figure S4), and the corresponding signal outputs at 5 min after injection (denoted as signal intensity) are summarized in Figure 3B. As clearly seen in Figure 3A,B, the DNA-coated MSS exhibited a significantly high sensitivity to water with ΔR/R~0.35 (Vout~44 mV; see also Equation (1)), while the sensitivity to other organic solvents was ca. 15–20 times lower, despite their hydrophilicity as well as water-miscibil-ity. Interestingly, the response to benzene, which has a planer structure and is known as a DNA intercalator, exhibited a similar intensity to those measured for other organic sol-vent vapors. According to the working principle of nanomechanical sensors in static mode operation, the signal output is obtained by the surface stress induced by mechanical de-formation [26]. Thus, the intercalation of benzene between the base pairs of DNA seemed to have less effect on mechanical deformation (i.e., expansion of DNA structure), resulting in a low signal response. We further investigated this remarkable response of the DNA receptor layers to wa-ter. The relative humidity (RH) was varied in the range of 0–80%, and the corresponding signal responses were measured (Figure 3C). The signal intensity rapidly increased in the range of 0% to 10% and then it gradually increased. It should be noted that the signal intensity even at 2% RH was 18.1 mV, significantly higher than those for all vapors of organic solvents at Pa/Po = 80%. Since the theoretical noise of the MSS is ca. 1 µV [36] and the experimentally observed noise was ca. 5 µV, the Limit of Detection (LoD) [41] was estimated as 1.7 × 10–5% RH. Compared to other humidity sensors [42], the DNA-based MSS exhibits high sensitivity to water (Table S1 in the supplementary materials). This re-markable selectivity and sensitivity of the DNA-coated MSS to water has a great potential for the detection of trace amounts of water in organic solvents.  Figure 3. Moisture-selective response of the DNA-coated MSS. (A) Signal responses to various va-pors at the concentration of Pa/Po = 80%. Signal responses to all vapors; water (black), water-miscible organic solvents (blue), and water-immiscible organic solvents (red). All responses are shown in Figure S4. (B) Signal intensities for each vapor. (C) Humidity-dependent responses in the range of 0–80% RH (black). Red plots indicate the FEA results. (D) Frequency shifts of DNA-coated QCM in the range of 0–90%RH. (E) Frequency shifts of DNA-coated QCM as a function of relative humidity. (F) Plausible mechanism of hydration-induced electrostatic repulsion. To further investigate the mechanism of the selectivity to water vapor, we estimated the hydration-induced strain by using a QCM, followed by a numerical simulation Figure 3. Moisture-selective response of the DNA-coated MSS. (A) Signal responses to various vaporsat the concentration of Pa/Po = 80%. Signal responses to all vapors; water (black), water-miscibleorganic solvents (blue), and water-immiscible organic solvents (red). All responses are shown inFigure S4. (B) Signal intensities for each vapor. (C) Humidity-dependent responses in the range of0–80% RH (black). Red plots indicate the FEA results. (D) Frequency shifts of DNA-coated QCM inthe range of 0–90%RH. (E) Frequency shifts of DNA-coated QCM as a function of relative humidity.(F) Plausible mechanism of hydration-induced electrostatic repulsion.We further investigated this remarkable response of the DNA receptor layers to water.The relative humidity (RH) was varied in the range of 0–80%, and the corresponding signalresponses were measured (Figure 3C). The signal intensity rapidly increased in the range of0% to 10% and then it gradually increased. It should be noted that the signal intensity evenat 2% RH was 18.1 mV, significantly higher than those for all vapors of organic solventsat Pa/Po = 80%. Since the theoretical noise of the MSS is ca. 1 µV [36] and the experi-mentally observed noise was ca. 5 µV, the Limit of Detection (LoD) [41] was estimated as1.7 × 10−5% RH. Compared to other humidity sensors [42], the DNA-based MSS exhibitshigh sensitivity to water (Table S1 in the supplementary materials). This remarkable selec-tivity and sensitivity of the DNA-coated MSS to water has a great potential for the detectionof trace amounts of water in organic solvents.To further investigate the mechanism of the selectivity to water vapor, we estimatedthe hydration-induced strain by using a QCM, followed by a numerical simulation throughFEA. The same volume of DNA solution was cast on a QCM. Frequency shifts weremeasured with varied relative humidity in the range from 0% RH to 90% RH (Figure 3D).The frequency shifts at 5 min after moisture injection are plotted in Figure 3E. As clearlyseen in comparison with Figure 3C,E, the trend of the QCM was different from the trendBiosensors 2022, 12, 1103 7 of 11of the MSS. The signal intensity of the MSS rapidly increased up to 10% RH, while thefrequency shift of the QCM exhibited linear correlation up to 70% RH, decreasing at therate of 0.53 Hz per % RH. According to Sauerbrey’s equation [43], we estimated the weightof the absorbed water molecules from the frequency shifts of the QCM. Assuming that thespecific volume of absorbed water in the DNA film does not change, the hydration-inducedstrain can be estimated as 2.5 × 10−5 strain per % RH. By using this calculated strain,the trend in the signal output was numerically simulated through FEA (Figure 3C, red).Compared to the FEA results as well as to the QCM trend, the humidity-dependent signalintensity of the DNA-coated MSS was different, especially at the lower relative humidity(i.e., up to 10% RH). In a DNA layer, where water is present, repulsive hydration forcesbetween phosphate groups have been reported [21,44]. These repulsive hydration forcesmay contribute to the mechanical deformation of the DNA film, resulting in its remarkablesensitivity to water.3.3. Detection of Trace Amounts of Water in Organic SolventsMost organic solvents are frequently contaminated with water. Such water, particu-larly trace amounts of water, has a great influence on chemical reactions [30,45]. Thus, thedetection and quantification of trace amounts of water in organic solvents are of crucial im-portance in chemistry and in the industry. Karl Fischer titration is a commonly used methodfor determining the water content based on coulometric and volumetric analyses [27–29],whereas it also has some drawbacks in terms of rapid and direct detection because of itstime-consuming sample preparation and need of specific equipment [46,47]. Althoughsome of the alternative approaches including colorimetry [31], fluorometry [30,31], elec-trochemistry [32], and nuclear magnetic resonance [33] have been reported, there are stillsome limitations such as low sensitivity, requirement of expensive equipment, and someinsufficiencies in probe materials. In contrast to the conventional Karl Fischer titrationas well as to other reported methods, the DNA-based nanomechanical sensors presentedin this study can provide an alternative method for detecting and quantifying such traceamounts of water.To explore the possibility of using the DNA-coated MSS for the quantification of traceamounts of water in organic solvents, we measured the vapors of water-contaminatedorganic solvents. We prepared the dehydrated organic solvents by dehydration withmolecular sieves. Subsequently, an aliquot of water was added to prepare a wide range ofwater concentrations up to 0.4 w/w% (i.e., 4000 ppm). Four water-miscible organic solventswere examined: THF, acetone, acetonitrile, and pyridine. The signal responses are depictedin Figure 4A and Figure S5. The signal intensity measured for each organic solvent clearlydepended on the concentration of water in the organic solvents in the measured range(Figure 4B), with a linear correlation for the low concentration range (Figure 4C). In the caseof THF, the DNA-coated MSS yielded an intensity of ca. 2 mV at a 12 ppm water content.Since the theoretical noise level of the MSS is 1 µV [36] and the experimental noise levelwas ca. 5 µV, the present system is capable of detecting water at the parts-per-billion (ppb)level in organic solvents (Table S2).Compared to Karl Fischer titration, the water content could be quantified in a shortertime, e.g., in 30 s after vapor injection (Figure S5C). Furthermore, in the case of THF, thesignal output at 2 s after vapor injection allowed us to quantify the water content, as shownin Figure 4D. More importantly, the DNA-coated MSS can be utilized for the quantificationof water in pyridine. Since the chemical reaction used in Karl Fischer titration typicallyrequires a base reagent (e.g., pyridine) [27–29], such solvents are generally impossible toquantify by Karl Fischer titration. Therefore, DNA-based nanomechanical sensors offer apromising alternative to Karl Fischer titration, providing a simple and rapid method fordetecting and quantifying trace water contents in various organic solvents.Biosensors 2022, 12, 1103 8 of 11Biosensors 2022, 12, x FOR PEER REVIEW 8 of 11  possible to quantify by Karl Fischer titration. Therefore, DNA-based nanomechanical sen-sors offer a promising alternative to Karl Fischer titration, providing a simple and rapid method for detecting and quantifying trace water contents in various organic solvents.  Figure 4. Quantification of trace amounts of water in various organic solvents. (A) Concentration-dependent signal responses to water in THF. The signal responses to other solvents are summarized in Figure S5. (B,C) Plots of signal intensities as a function of water concentration in the ranges of 0–0.4% (B) and 0–600 ppm (C) with different organic solvents: THF (black); acetone (red); acetonitrile (blue); and pyridine (orange). (D) Plot of signal output to THF, 2 s after injection. 3.4. Reproducibility of the DNA-Based MSS In practical applications, the reproducibility of the sensing performance is an im-portant factor. To investigate the stability and reproducibility of the DNA-based MSS, we repeated the sensing experiments to detect water vapor and trace amounts of water in an organic solvent. Water vapor at 80% RH and vapor of 100 ppm water in THF were meas-ured for 50 cycles. As can be seen in Figure S6, the signal intensities in the detection of water vapor and trace amounts of water were 45.97 ± 0.03 mV and 6.81 ± 0.34 mV, respec-tively. These results clearly indicated that the DNA-based MSS has high stability and re-producibility when detecting the vapors of water as well as trace amounts of water in chemicals, suggesting its practical applicability. 4. Conclusions We demonstrated that a DNA-coated MSS exhibited high sensitivity and selectivity to water, leading to the facile and rapid detection of trace amounts of water in organic solvents. Taking advantage of nanomechanical sensors in the static mode operation, the MSS can efficiently and selectively detect the mechanical deformation of a DNA layer in-duced by water absorption. This high sensitivity and selectivity enable DNA-coated MSS to detect trace amounts of water in organic solvents as low as 12 ppm (i.e., 0.0012 w/w%). Although the mechanism of water sensitivity needs further investigation, given the ro-bustness and compactness of the MSS sensing system [26] and its high stability and repro-ducibility, this study provides a simple and rapid method for the detection and quantifi-cation of trace amounts of water in organic solvents as an alternative to the conventional Karl Fischer titration. Furthermore, this method can provide a promising sensing platform for the on-site detection of water, with a large variety of potential applications in various fields including chemistry, food, environment, healthcare, and medicine.   Figure 4. Quantification of trace amounts of water in various organic solvents. (A) Concentration-dependent signal responses to water in THF. The signal responses to other solvents are summarizedin Figure S5. (B,C) Plots of signal intensities as a function of water concentration in the ranges of0–0.4% (B) and 0–600 ppm (C) with different organic solvents: THF (black); acetone (red); acetonitrile(blue); and pyridine (orange). (D) Plot of signal output to THF, 2 s after injection.3.4. Reproducibility of the DNA-Based MSSIn practical applications, the reproducibility of the sensing performance is an impor-tant factor. To investigate the stability and reproducibility of the DNA-based MSS, werepeated the sensing experiments to detect water vapor and trace amounts of water inan organic solvent. Water vapor at 80% RH and vapor of 100 ppm water in THF weremeasured for 50 cycles. As can be seen in Figure S6, the signal intensities in the detectionof water vapor and trace amounts of water were 45.97 ± 0.03 mV and 6.81 ± 0.34 mV,respectively. These results clearly indicated that the DNA-based MSS has high stability andreproducibility when detecting the vapors of water as well as trace amounts of water inchemicals, suggesting its practical applicability.4. ConclusionsWe demonstrated that a DNA-coated MSS exhibited high sensitivity and selectivity towater, leading to the facile and rapid detection of trace amounts of water in organic solvents.Taking advantage of nanomechanical sensors in the static mode operation, the MSS canefficiently and selectively detect the mechanical deformation of a DNA layer induced bywater absorption. This high sensitivity and selectivity enable DNA-coated MSS to detecttrace amounts of water in organic solvents as low as 12 ppm (i.e., 0.0012 w/w%). Althoughthe mechanism of water sensitivity needs further investigation, given the robustness andcompactness of the MSS sensing system [26] and its high stability and reproducibility, thisstudy provides a simple and rapid method for the detection and quantification of traceamounts of water in organic solvents as an alternative to the conventional Karl Fischertitration. Furthermore, this method can provide a promising sensing platform for theon-site detection of water, with a large variety of potential applications in various fieldsincluding chemistry, food, environment, healthcare, and medicine.5. PatentsT.M., K.M., T.Y., G.Y., and K.A. are inventors, Japanese patent application number2022-148684, submitted by National Institute for Materials Science (NIMS).Biosensors 2022, 12, 1103 9 of 11Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/bios12121103/s1, Figure S1: Sensing sequence; Figure S2: Surfaceprofile of the DNA film on the MSS; Figure S3: Electrophoresis; Figure S4: Signal responses to organicsolvent vapors; Figure S5: Signal responses to water in organic solvents; Figure S6: Reproducibility;Table S1: Limit of Detection to humidity [34,36,42,48–61]; Table S2: Limit of Detection to trace amountsof water in organic solvents.Author Contributions: Conceptualization, K.M., T.Y., G.Y. and K.A.; methodology, T.M. and K.M.;software, G.Y.; validation, K.M.; formal analysis, T.M.; investigation, T.M. and K.M.; resources,T.Y.; data curation, T.M. and K.M.; writing—original draft preparation, T.M. and K.M.; writing—review and editing, T.Y., G.Y. and K.A.; visualization, T.M. and K.M.; supervision, K.A.; projectadministration, K.A.; funding acquisition, K.M., T.Y., G.Y. and K.A. All authors have read and agreedto the published version of the manuscript.Funding: This study was financially supported by a Grant-in-Aid for Scientific Research (A), MEXT,Japan (No. 18H04168); Grant-in-Aid for Scientific Research (B), MEXT, Japan (No. 21H01971);Grant-in-Aid for Scientific Research (C), MEXT, Japan (No. 20K05345); Grant-in-Aid for ScientificResearch (C), MEXT, Japan (No. 22K05324); Grant-in-Aid for Exploratory Research, MEXT, Japan(No. 21K18859); Grant-in-Aid for Challenging Research (Pioneering) (No. 20K20554); Fostering JointInternational Research (B), MEXT, Japan (No. JP19KK0141); Support for Pioneering Research Instituteby the Next Generations (SPRING), JST, MEXT, Japan (No. JPMJSP2108); the Public/Private R&DInvestment Strategic Expansion Program (PRISM), Cabinet Office, Japan; and Center for FunctionalSensor & Actuator (CFSN), NIMS.Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.Data Availability Statement: Not applicable.Acknowledgments: T.M. thanks the Support for Pioneering Research Institute by the Next Genera-tions (SPRING) program, JST, MEXT, Japan (No. JPMJSP2108). 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