Calibration-free Measurements Research Articles

Mid-infrared absorption spectroscopy for in-situ and spatially resolved measurements of nitric oxide in premixed ammonia-methane co-fired flames.

Laser absorption spectroscopy (LAS) is a well-established measurement technique for quantitative chemical speciation in a combustion environment. However, in-situ LAS measurement of nitric oxide (NO) in ammonia flames has never been reported in the literature. This is despite the community's recent strong interest in carbon-neutral ammonia combustion and the associated NO formation problem. In this work, we demonstrate the development and validation of a mid-infrared laser absorption sensor for in-situ measurements of NO formation and evolution in premixed ammonia and ammonia-methane cofired flames. To achieve calibration-free and interference-free measurements, the sensor exploits the NO absorption feature near 1900.07 cm-1 using the techniques of both direct absorption spectroscopy and wavelength modulation spectroscopy. Special efforts were given to address the thermochemical non-uniformity along the light path which was shown to have notable effects on measurement accuracy. Detailed computational fluid dynamics modeling on the flame structure was performed along with theoretical spectral simulation to assist in the treatment of the non-uniformity effects. Comprehensive measurements were then performed in flames with different ammonia proportions and equivalence ratios, with results compared to data from probe sampling methods and kinetic modeling. The present work is the first demonstration of an in-situ mid-infrared LAS sensor for quantitative and spatially resolved NO measurement in ammonia flames.

An ML-Enhanced Laser-Based Methane Slip Sensor Using Wavelength Modulation Spectroscopy.

Natural gas (NG) is a promising alternative to diesel for sustainable transport, potentially reducing GHG and air quality emissions significantly. However, the GHG benefits hinge on managing methane slip, the unburned methane in the exhaust of NG engines, which carries a significant global warming potential. The CH4 slip from NG engines is highly dependent on engine type and operation, and effective greenhouse gas emission mitigation requires that the actual operation of real-world engines is monitored. This requires suitable instrumentation for online robust CH4 measurement in engine exhaust. Traditional methane slip measurement methods need frequent calibration, may not be suited to dynamic operational conditions, carry significant costs, or require expert users. Furthermore, the significant computational demands associated with calibration-free spectroscopic methods and the prevalent noise uncertainty underscore the urgent requirement for innovative sensors. These sensors must not only respond rapidly but also have low uncertainty in their readings. This paper presents a machine learning (ML)-enhanced, laser-based methane slip sensor using wavelength modulation spectroscopy (WMS) for rapid, accurate, and calibration-free CH4 measurements for application in the exhaust of NG engines. The sensor utilizes a distributed feedback (DFB) laser diode emitting around 1.65 μm propagated through a multipass optical cell. An ML-based approach is used to invert the recorded WMS signal, which reduces computational cost and uncertainty due to noise vulnerabilities inherent in traditional measurement inversion approaches. A Gaussian process regression (GPR) model, trained on measured and simulated WMS signals, was selected for its high predictive accuracy, where it achieved a mean absolute percent error (MAPE) of 0.24%. For exhaust measurement on an in-use natural gas marine vessel, a mean absolute difference of 3.95% was observed, relative to simultaneous reference Fourier transform infrared spectroscopy measurements. The ML-based WMS inversion system marks a significant advancement in methane slip measurement, offering real-time monitoring capabilities with reduced computational demands. Its development supports the realization of NG environmental benefits for transport by providing accurate CH4 slip data, which are essential for engine performance optimization, regulatory adherence, and sustainable policy decisions.

Dual-angle light scattering spectroscopy for calibration-free measurements of scatterer suspensions

It can be difficult to employ optical techniques for analyzing biological structures smaller than or comparable to the wavelength of light, such as extracellular vesicles or some types of bacteria. Biological light scattering spectroscopy (LSS), developed to address this problem, has been successfully used for characterizing tissue on cellular and subcellular scales. At the same time, calibration with a reference sample of known optical properties can complicate LSS measurements. In this Letter, we present dual-angle LSS (daLSS), which is designed for calibration-free measurements of scatterer suspensions. It employs measurements of a sample at two distinct angles, which then allows system effects to be removed entirely. Not only does daLSS simplify and speed up the measurement procedure, but it also makes spectra more reproducible, an important feature for diagnostic techniques. We validated the technique by accurately reconstructing the sizes of polystyrene microspheres with diameters less than 100 nm and then demonstrated that not only are the daLSS spectra of several common bacteria strains easily distinguishable but they are also highly reproducible.

Calibration-free thickness and temperature measurement of oil films using broadband near-infrared absorption spectroscopy

Abstract This paper assesses broadband absorption spectroscopy for calibration-free, single-ended thickness and temperature measurements of thin liquid films with a free interface using spectrally resolved intensity measurements. The spectroscopic properties of the liquid in question, here an oil lubricant, are characterized using a Fourier transform infrared spectrometer. This data forms the basis for a measurement model that is used to infer the thickness and temperature of films wetting metal surfaces based on broadband spectral transmission measurements. The technique is demonstrated by inferring the properties of static films of known properties as well as free films on a rotating metal disk.

Calibration-free heterodyne phase-sensitive dispersion spectroscopy: quantitative gas sensing and recovery of absorption spectra.

Heterodyne phase-sensitive dispersion spectroscopy (HPSDS) is a quantitative non-intrusive gas sensing technique based on the determination of the refractive index of the target gas in the vicinity of an absorption transition. Since the phase instead of the intensity of the probing laser light is targeted, the technique boasts the advantage of being normalization-free. It is thus largely immune to laser intensity fluctuations due to either system instability or ambient interferences. Previous HPSDS-based sensors typically require calibration using standard mixtures to establish a look-up table between the measured phase signal and gas concentrations, which is both cumbersome and problematic when there are significant compositional variations between the calibration standards and the target gas. In this work, we present a robust and generic technique that addresses this issue with a successful realization of fully calibration-free measurements. Spectral-fitting to the entire dispersion spectra with free variables related to transition linecenter, broadening width, and integrated absorbance were used to eliminate the effects of unknown spectral broadening coefficients. What we believe to be a novel analytical model was proposed to unify both direct injection-current dithering-based HPSDS that includes simultaneous frequency/intensity modulation, and the external electro-optic modulator (EOM) modulation-based HPSDS with a non-ideal linear response of EOM. The proposed technique was first validated via numerical experiment to determine the gas concentration and the recovery of the absorption profiles. Actual experiments were subsequently performed for the measurement of CH4 near 1.65 µm, N2O near 4.46 µm, and NO near 5.26 µm, collectively demonstrating the capability of the technique for both near- and mid-infrared lasers with diverse modulation characteristics. Further demonstrations were performed to measure NH3 concentrations at elevated temperatures through the fitting of the multiple dispersion spectra near 9.06 µm. The robust iterative spectral-fitting strategy and the measurement accuracies confirm the robustness of the proposed calibration-free (CF) HPSDS technique for quantitative gas sensing.

Detailed analysis of the R1f/ΔI1 WMS technique and demonstration of significantly higher detection sensitivity compared to 2f WMS for calibration-free trace gas sensing.

Recognizing that wavelength modulation spectroscopy (WMS) is particularly important in the development of high-sensitivity gas sensing systems, this paper presents a detailed analysis of the R 1f /Δ I 1 WMS technique that has recently been successfully demonstrated for calibration-free measurements of the parameters that support detecting multiple gases under challenging conditions. In this approach, the magnitude of the 1f WMS signal (R 1f ) was normalized by using the laser's linear intensity modulation (Δ I 1) to obtain the quantity R 1f /Δ I 1 that is shown to be unaffected by large variations in R 1f itself due to the variations in the intensity of the received light. In this paper, different simulations have been used to explain the approach taken and the advantages that it shows. A 40mW, 1531.52nm near-infrared distributed feedback (DFB) semiconductor laser was used to extract the mole fraction of acetylene in a single-pass configuration. The work has shown a detection sensitivity of 0.32ppm for 28cm (0.089ppm-m) with an optimum integration time of 58s. The detection limit achieved has been shown to be better than the value of 1.53ppm (0.428ppm-m) for R 2f WMS by a factor of 4.7, which is a significant improvement.

Employing a Redox Reporter-Modified Self-Assembly Monolayer in Electrochemical Aptamer-Based Sensors to Enable Calibration-Free Measurements

Electrochemical aptamer-based (E-AB) sensors suffer from sensor-to-sensor signal variations due to the variation in the total number of probes immobilized on the sensor surface, the effective working area, and the heterogeneity properties of the electrode surface, thus requiring a calibration step prior to each measurement. This is impractical, if not possible, for some cases, e.g., in a complex matrix including blood samples. In response, we propose a calibration-free approach to achieve the measurement of biorelevant small-molecule and protein analytes. Specifically, we employed one reporter labeled onto an aptamer (e.g., methylene blue) for redox signaling, and the other reporter (e.g., ferrocene) was modified onto a self-assembly monolayer as a reference signal. By taking the ratio of the two signals, we achieved a much improved baseline stability and sensor-to-sensor reproducibility, which allows the calibration-free measurement of the analysis of the respective targets, including doxorubicin, vancomycin, and thrombin in both simple buffer and even directly complex samples including serum and whole blood.

Simultaneous Measurement of Multiparameter of Diesel Engine Exhaust Based on Mid-Infrared Laser Absorption Spectroscopy

Exhaust parameters of an engine carry vital information that can help to understand its transient duty cycles. We developed a mid-infrared wavelength modulation spectroscopic sensor for simultaneous measurement of exhaust temperature, pressure, and NO and NO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> concentrations by using two quantum cascade lasers (QCLs) for the first time. The dual-channel signals were demodulated and normalized respectively with a specially designed digital lock-in amplifier, achieving calibration-free measurements to adapt to the harsh and variable working conditions of diesel engines. Two-line thermometry was performed using a newly selected spectral line pair of NO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> (1629.85 and 1630.33 cm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">-1</sup> ). Simultaneous measurement of the quadruple parameter of diesel exhaust, i.e. temperature, pressure, and NO and NO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> concentrations, was first achieved using only three spectral lines and a developed multi-parameter inversion algorithm. The feasibility of the sensor was demonstrated in field tests in an in-service vehicle. Exhaust temperature, pressure, and NO and NO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> concentrations were obtained continuously with 0.1 s time intervals, which clearly show these parameters in the transition and steady stages of the engine, respectively. The method and results can be used to evaluate the engine operation performance and combustion diagnostics.

Distance Measurement Using mmWave Radar: Micron Accuracy at Medium Range

This work provides a proof-of-concept for a linear-position sensor using an ultrawideband (UWB) frequency-modulated continuous wave (FMCW) radar system operating at 126–182 GHz. It is the first work to show environmental compensated and calibration-free distance measurements with micron accuracy at medium range using millimeter-wave (mmWave) radar technology. We addressed hardware imperfections, parameter estimation, and the free-space path, i.e., refractive index and near-field effects. The proposed signal processing chain is robust to interference and of low computational cost. Experiments reveal a systematic error of <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\pm 1~\mu \text{m}$ </tex-math></inline-formula> over 4.8 m (0.8–5.6 m), and a random error at a minimum of 30 nm, providing very high sensitivity.

Kinetic characterization of SPR-based biomarker assays enables quality control, calibration free measurements and robust optimization for clinical application

ABSTRACT: Biomarker measurements are essential for the early diagnosis of complex diseases. However, many current biomarker assays lack sensitivity and multiplexing capacity, work in a narrow detection range and importantly lack real time quality control opportunities, which hampers clinical translation. In this paper, we demonstrate a toolbox to kinetically characterize a biomarker measurement assay using Surface Plasmon Resonance imaging (SPRi) with ample opportunities for real time quality control by exploiting quantitative descriptions of the various biomolecular interactions. We show an accurate prediction of SPRi measurements at both low and high concentrations of various analytes with deviations <5% between actual measurements and predicted measurement. The biphasic binding sites model was accurate for fitting the experimental curves and enables optimal detection of heterophilic antibodies, cross-reactivity, spotting irregularities and/or other confounders. The toolbox can also be used to create a (simulated) calibration curve, enabling calibration-free measurements with good recovery, it allows for easy assay optimizations, and could help bridge the gap to bring new biomarker assays to the clinic.

Concept of the "Universal Slope": Toward Substantially Shorter Decentralized Insulin Immunoassays.

Decentralized sensing of analytes in remote locations is today a reality. However, the number of measurable analytes remains limited, mainly due to the requirement for time-consuming successive standard additions calibration used to address matrix effects and resulting in greatly delayed results, along with more complex and costly operation. This is particularly challenging in commonly used immunoassays of key biomarkers that typically require from 60 to 90 min for quantitation based on two standard additions, hence hindering their implementation for rapid and routine diagnostic applications, such as decentralized point-of-care (POC) insulin testing. In this work we have developed and demonstrated the theoretical framework for establishing a universal slope for direct calibration-free POC insulin immunoassays in serum samples using an electrochemical biosensor (developed originally for extended calibration by standard additions). The universal slope is presented as an averaged slope constant, relying on 68 standard additions-based insulin determinations in human sera. This new quantitative analysis approach offers reliable sample measurement without successive standard additions, leading to a dramatically simplified and faster assay (30 min vs 90 min when using 2 standard additions) and greatly reduced costs, without compromising the analytical performance while significantly reducing the analyses costs. The substantial improvements associated with the new universal slope concept have been demonstrated successfully for calibration-free measurements of serum insulin in 30 samples from individuals with type 1 diabetes using meticulous statistical analysis, supporting the prospects of applying this immunoassay protocol to routine decentralized POC insulin testing.

High-frame-rate A-mode ultrasound for calibration-free cuffless carotid pressure: feasibility study using lower body negative pressure intervention

Purpose Existing technologies to measure central blood pressure (CBP) intrinsically depend on peripheral pressure or calibration models derived from it. Pharmacological or physiological interventions yielding different central and peripheral responses compromise the accuracy of such methods. We present a high-frame-rate ultrasound technology for cuffless and calibration-free evaluation of BP from the carotid artery. The system uses a pair of single-element ultrasound transducers to capture the arterial diameter and local pulse wave velocity (PWV) for the evaluation of beat-by-beat BP employing a novel biomechanical model. Materials and methods System’s functionality assessment was conducted on eight male subjects (26 ± 4 years, normotensive and no history of cardiovascular risks) by perturbing pressure via short-term moderate lower body negative pressure (LBNP) intervention (−40 mmHg for 1 min). The ability of the system to capture dynamic responses of carotid pressure to LBNP was investigated and compared against the responses of peripheral pressure measured using a continuous BP monitor. Results While the carotid pressure manifested trends similar to finger measurements during LBNP, the system also captured the differential carotid-to-peripheral pressure response, which corroborates the literature. The carotid diastolic and mean pressures agreed with the finger pressures (limits-of-agreement within ±7 mmHg) and exhibited acceptable uncertainty (mean absolute errors were 2.4 ± 3.5 and 2.6 ± 4.0 mmHg, respectively). Concurrent to the literature, the carotid systolic and pulse pressures (PPs) were significantly lower than those of the finger pressures by 11.1 ± 9.4 and 11.3 ± 8.2 mmHg, respectively (p < .0001). Conclusions The study demonstrated the method’s potential for providing cuffless and calibration-free pressure measurements while reliably capturing the physiological aspects, such as PP amplification and dynamic pressure responses to intervention.

Real-Time Tunable Dynamic Range for Calibration-Free Biomolecular Measurements with a Temperature-Modulated Electrochemical Aptamer-Based Sensor in an Unprocessed Actual Sample.

The sensing technologies for monitoring molecular analytes in biological fluids with high frequency and in real time could enable a broad range of applications in personalized healthcare and clinical diagnosis. However, due to the limited dynamic range (less than 81-fold), real-time analysis of biomolecular concentration varying over multiple orders of magnitude is a severe challenge faced by this class of analytical platforms. For the first time, we describe here that temperature-modulated electrochemical aptamer-based sensors with a dynamically adjustable calibration-free detection window could enable continuous, real-time, and accurate response for the several-hundredfold target concentration changes in unprocessed actual samples. Specifically, we could regulate the electrode surface temperature of sensors to obtain the corresponding dynamic range because of the temperature-dependent affinity variations. This temperature modulation method relies on an alternate hot and cold electrode reported by our group, whose surface could actively be heated and cooled without the need for altering ambient temperature, thus likewise applying for the flowing system. We then performed dual-frequency calibration-free measurements at different interface temperatures, thus achieving an extended detection window from 25 to 2500 μM for procaine in undiluted urine, 1-500 μM for adenosine triphosphate, and 5-2000 μM for adenosine in undiluted serum. The resulting sensor architecture could drastically expand the real-time response range accessible to these continuous, reagent-less biosensors.

QCL-Based Open-Path, Single-Pass Measurement of Ambient Carbon Monoxide Using R 1f /ΔI₁ WMS

This letter outlines a new robust wavelength modulation spectroscopy technique to achieve calibration-free measurements. The technique normalizes the magnitude of the <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$1^{st}$ </tex-math></inline-formula> harmonic signal by the amplitude of the laser’s linear intensity modulation, and uses the fact that the ratio of the intensity to the amplitude of the linear intensity modulation remains invariant at every point within a system and over time despite significant and unpredictable variations in the individual quantities. The technique is demonstrated using a mid-infrared quantum cascade laser for open-path measurements of ambient carbon monoxide with a single-pass detection limit of 12 ppb-m. The method is simple to implement and performs robustly for long durations of outdoor measurements.

Ultrafast-laser-absorption spectroscopy in the mid-infrared for single-shot, calibration-free temperature and species measurements in low- and high-pressure combustion gases.

This manuscript presents an ultrafast-laser-absorption-spectroscopy (ULAS) diagnostic capable of providing calibration-free, single-shot measurements of temperature and CO at 5 kHz in combustion gases at low and high pressures. Additionally, this diagnostic was extended to provide 1D, single-shot measurements of temperature and CO in a propellant flame. A detailed description of the spectral-fitting routine, data-processing procedures, and determination of the instrument response function are also presented. The accuracy of the diagnostic was validated at 1000 K and pressures up to 40 bar in a heated-gas cell before being applied to characterize the spatiotemporal evolution of temperature and CO in AP-HTPB and AP-HTPB-aluminum propellant flames at pressures between 1 and 40 bar. The results presented here demonstrate that ULAS in the mid-IR can provide high-fidelity, calibration-free measurements of gas properties with sub-nanosecond time resolution in harsh, high-pressure combustion environments representative of rocket motors.

Application of a novel ionic-liquid-based membrane reference electrode with inorganic insertion material paste to a calibration-free all-solid-state ion sensor chip

We propose an all-solid ionic-liquid-based membrane reference electrode (IL-RE) with the inner solid electrode coated with insertion material paste. This material paste contains an inorganic insertion material (Na0.33MnO2) and a solid electrolyte (β’’-alumina). The inorganic insertion material, Na0.33MnO2, in the paste exhibits a redox reaction accompanied by the disinsertion/insertion of Na+ and acts as an ion-to-electron transducer. Furthermore, β’’-alumina in the paste acts as a supply source of Na+ for Na0.33MnO2. The insertion material paste stabilizes the phase boundary potential of the inner solid electrode in the IL-RE, even if the IL membrane contains no inserted ions (Na+). The fabricated IL-RE exhibited high device-to-device reproducibility (the standard deviation of the IL-RE potential: ± 0.6 mV, 8 sensors). The IL-RE was integrated into a K+ sensor chip with the all-solid K+-ion selective electrode (K+-ISE) for calibration-free measurements. The K+ sensor chip was designed to prevent the effect of the IL eluted from the IL-RE on K+-ISE potential by optimizing IL concentration in the IL membrane and distance between IL-RE and K+-ISE. The fabricated K+ sensor chips exhibited a very high device-to-device potential reproducibility of ± 1.3 mV in KCl solutions and ± 2.2 mV even in blood serum samples. This ion sensor chip realized calibration-free measurement as disposable sensors and showed the possibility of facilitating mass production.

MicroPulse DIAL (MPD) – a diode-laser-based lidar architecture for quantitative atmospheric profiling

Abstract. Continuous water vapor and temperature profiles are critically needed for improved understanding of the lower atmosphere and potential advances in weather forecasting skill. Ground-based, national-scale profiling networks are part of a suite of instruments to provide such observations; however, the technological method must be cost-effective and quantitative. We have been developing an active remote sensing technology based on a diode-laser-based lidar technology to address this observational need. Narrowband, high-spectral-fidelity diode lasers enable accurate and calibration-free measurements requiring a minimal set of assumptions based on direct absorption (Beer–Lambert law) and a ratio of two signals. These well-proven quantitative methods are known as differential absorption lidar (DIAL) and high-spectral-resolution lidar (HSRL). This diode-laser-based architecture, characterized by less powerful laser transmitters than those historically used for atmospheric studies, can be made eye-safe and robust. Nevertheless, it also requires solar background suppression techniques such as narrow-field-of-view receivers with an ultra-narrow bandpass to observe individual photons backscattered from the atmosphere. We discuss this diode-laser-based lidar architecture's latest generation and analyze how it addresses a national-scale profiling network's need to provide continuous thermodynamic observations. The work presented focuses on general architecture changes that pertain to both the water vapor and the temperature profiling capabilities of the MicroPulse DIAL (MPD). However, the specific subcomponent testing and instrument validation presented are for the water vapor measurements only. A fiber-coupled seed laser transmitter optimization is performed and shown to meet all of the requirements for the DIAL technique. Further improvements – such as a fiber-coupled near-range receiver, the ability to perform quality control via automatic receiver scanning, advanced multi-channel scalar capabilities, and advanced processing techniques – are discussed. These new developments increase narrowband DIAL technology readiness and are shown to allow higher-quality water vapor measurements closer to the surface via preliminary intercomparisons within the MPD network itself and with radiosondes.

Towards an Optical Gas Standard for Traceable Calibration-Free and Direct NO2 Concentration Measurements

We report a direct tunable diode laser absorption spectroscopy (dTDLAS) instrument developed for NO2 concentration measurements without chemical pre-conversion, operated as an Optical Gas Standard (OGS). An OGS is a dTDLAS instrument that can deliver gas species amount fractions (concentrations), without any previous or routine calibration, which are directly traceable to the international system of units (SI). Here, we report NO2 amount fraction quantification in the range of 100–1000 µmol/mol to demonstrate the current capability of the instrument as an OGS for car exhaust gas application. Nitrogen dioxide amount fraction results delivered by the instrument are in good agreement with certified values of reference gas mixtures, validating the capability of the dTDLAS-OGS for calibration-free NO2 measurements. As opposed to the standard reference method (SRM) based on chemiluminescence detection (CLD) where NO2 is indirectly measured after conversion to NO, titration with O3 and the detection of the resulting fluorescence, a dTDLAS-OGS instrument has the benefit of directly measuring NO2 without distorting or delaying conversion processes. Therefore, it complements the SRM and can perform fast and traceable measurements, and side-by-side calibrations of other NO2 gas analyzers operating in the field. The relative standard uncertainty of the NO2 results reported in this paper is 5.1% (k = 1, which is dominated (98%) by the NO2 line strength), the repeatability of the results at 982.6 µmol/mol is 0.1%, the response time of the instrument is 0.5 s, and the detection limit is 825 nmol/mol at a time resolution of 86 s.

Easy-to-Make Capillary-Based Reference Electrodes with Controlled, Pressure-Driven Electrolyte Flow.

As solid-contact potentiometric sensors based on novel materials have reached exceptional stabilities with drifts in the low μV/h range and long-term and calibration-free potentiometric measurements gain more and more attention, reference electrode designs that used to be satisfactory for most users do not satisfy the needs of new challenging applications. It is important that the interface between a reference electrode and the sample, often provided by a salt bridge, remains constant in ion composition over time. Excessive restriction of the flow of the bridge electrolyte, e.g., by using nanoporous frits or gelled reference electrolyte solutions, can result in contamination of the salt bridge with sample components and depletion of the reference electrolyte by diffusion into samples. This can be avoided by using salt bridges that flow freely into the sample. However, commonly used reference electrodes with free-flowing junctions often suffer either from experimental difficulties in assuring a minimum flow rate or from excessive flow rates that require frequent replenishing of the bridge electrolyte. To this end, we developed a reference electrode that contains a concentrated electrolyte contacting samples through a 10.2 μm capillary. By applying a minimal pressure of 10.0 kPa, a flow rate of 100 nL/h is achieved. This maintains a constant liquid junction potential at the interface with the sample and avoids contamination of the reference electrode, as evidenced by a potential stability of 6 ± 3 μV/h over 21 days. With such a minimal flow rate, there is no need to refill the reference electrode electrolyte for years.

Hybridization Chain Reaction-Amplified Electrochemical DNA-Based Sensors Enable Calibration-Free Measurements of Nucleic Acids Directly in Whole Blood.

Hybridization chain reaction (HCR) amplification strategy has been extensively explored for the application of electrochemical DNA-based sensors. Despite the enhancement in its sensitivity using the HCR, such sensor platform exhibited significant sensor-to-sensor variations in current due to variations in probe counts and lengths. To circumvent this, we are developing here a calibration-free "O-N" approach to generate a ratiometric, unitless value that is independent of these variations. Specifically, this approach employs two types of redox reporters, denoted as "One reporter" and "N reporters", with the former attached on the capture DNA and the latter on H1 and H2 strands. By optimizing the attachment sites of these reporters onto DNA strands, we demonstrate a significantly enhanced sensitivity of such sensor platform by four orders of magnitude, achieving accurate, calibration-free measurement of nucleic acids including ctDNA directly in undiluted whole blood without the requirement to calibrate each individual sensor.