Small Sample Volumes Research Articles

Concentration of strontium and barium by co-precipitation with organic collectors and their x-ray fluorescence determination

The concentration of strontium and barium in the form of complexes with 11 organic reagents by co-precipitation with organic co-precipitants was studied for their subsequent determination by the X-ray fluorescence method. The most effective systems were those including reagents from the group of bisazo-substituted chromotropic acids—Nithchromazo and Chlorophosphonazo III. The complexes of these metals were almost quantitatively co-precipitated in the form of associates with the cations of the Brilliant Green dye when the collector was an associate of an excess of the analytical reagent with the cations of this dye. It was shown that the additional use of polyvinyl butyral as an indifferent co-precipitant allows not only the almost complete extraction of these elements from solutions but also the preparation of emitter concentrates suitable for X-ray fluorescence measurements using the background standard technique. The high efficiency allows reaching very low detection limits (IUPAC): 0.03 µg/mL for Sr and 0.19 µg/mL for Ba, even when working with small-volume samples.

Electrochemical immunosensing of Crohn's disease biomarkers using diazonium salt-grafted crystalline nanocellulose/carbon nanotube-modified electrodes.

Thefirst dual immunosensoris reportedfor the determination of IL-12 and IL-23, two relevant biomarkers of Crohn's disease (CD). The strategy relies on the selective capture of the targets by the respective antibodies which were covalently immobilized onto SPCEs modified with crystalline nanocellulose (CNC) and multi-walled carbon nanotubes (MWCNTs) followed by conjugation with a detector antibody labelled with poly-HRP-Strept and amperometric transduction using the H2O2/HQ system. The combination of CNC, a nanomaterial scarcely exploited in immunosensing, with MWCNTs enables the preparation of a novel dual immunosensor for the determination of CD biomarkers in clinical samples, including faeces. The developed bioplatform exhibits a high reproducibility and selectivity allowing the simultaneous determination of IL-12 and IL-23 using a smaller sample volume and in a much shorter assay time than those reported for each target with commercial ELISA kits. The dual immunosensor was successfully applied to the analysis of raw serum and faeces spiked with IL-12 and IL-23 at the expected levels in patients with severe CD.

Utilization and validation of dried blood spot-based metabolomics in plasma-derived diagnostic models

Metabolomics has rapidly advanced in life sciences, enhancing our understanding of physiological conditions. Plasma stands out as a predominant sample type in metabolomics studies. Dried blood spot (DBS) has been recognized as a valuable strategy due to its unique characteristics, such as minimal invasiveness, small sample volume, and easy transport. Most large cohort studies construct the diagnostic model using plasma-based metabolomics rather than DBS. However, the comparability of metabolite measurements between DBS and plasma samples, as well as the integration of DBS-based metabolomics into established plasma-derived diagnostic models, remains unclear. We collected plasma and DBS samples from the same 12 healthy controls under fasting and non-fasting conditions and performed ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS)-based metabolomics. We identified 277 metabolites present in both plasma and DBS samples, with 229 (82.7 %) showing a strong correlation between the two sample types. Furthermore, pre-processing datasets from plasma and DBS samples before combining them effectively diminished inconsistencies between plasma- and DBS-based metabolomics without compromising the inherent classification within the data. For the clinical application, the plasma samples were obtained from the gastric cancer patients (n = 204) and the healthy controls (n = 128) to serve as the training cohorts. Additionally, DBS samples were collected from different participants of gastric cancer (n = 19) and healthy controls (n = 12) to validate the diagnostic model. Finally, DBS-based metabolomics were integrated into established plasma-derived gastric cancer diagnostic models, yielding an area under the ROC curve (AUC) of 0.934. Overall, incorporating DBS-based metabolomics into an established plasma-derived diagnostic model holds promise, offering valuable opportunities and insights for diagnostic research and clinical practice.

Small volume solid phase extraction method for comprehensive analysis of neonicotinoids, their metabolites, and related pesticides in water

Neonicotinoid insecticides (NEOs) such as clothianidin, imidacloprid, and thiamethoxam are used worldwide. The occurrence of their degradates, for instance, clothianidin-n-desmethyl (CLO-N-DES), clothianidin-urea (CLOU), imidacloprid urea (IMIU) and olefin (IMIO), as well as thiamethoxam urea (THXU), have seldom been documented in water due to the lack of a sensitive analytical method. In this study, a method only requiring 12 mL of water sample was developed and validated to quantify 8 NEOs, 13 metabolites, and 3 related insecticides using solid phase extraction (SPE) coupled with HPLC-MS/MS. The method demonstrated good linearity (r2 > 0.99), with limits of detection (LOD) ranging from 0.16 to 1.21 ng/L and limits of quantification (LOQ) from 0.54 to 4.03 ng/L in water samples. Validation showed accuracy between 70 and 130 % and precision below 15 % for most analytes. The method's performance was comparable to, or better than, existing methods, with the advantage of requiring much smaller sample volumes. Using this method, we monitored the occurrence and seasonal variability of NEOs and their metabolites in various surface water and groundwaters matrices from across Iowa. For example, analysis of water samples from private wells across three Iowa counties detected several NEOs, with notable findings including the first detection of flupyradifurone (FLU) in Iowa well water. Surface water analysis from five locations revealed frequent detection of NEOs and their metabolites, with some concentrations exceeding U.S. EPA chronic toxicity benchmarks for freshwater invertebrates. In addition, this is the first study to demonstrate the occurrence of CLO-N-DES, CLOU, and THX-U in US surface water. The study helps advance analytical methods for NEOs and their metabolites while also highlighting their widespread occurrence in Iowa waters and associated ecological risks, emphasizing the need for more comprehensive monitoring of these compounds.

Exosomal Micro RNA Isolation in White-Tailed Deer (Odocoileus virginianus) for Diagnostic Biomarker Discovery.

Molecular approaches are becoming more prevalent for the diagnosis of neurodegenerative diseases in human medicine and can be extended to diagnosis of wildlife diseases such as chronic wasting disease and other prion diseases. These diseases have been associated with exosome-bound molecular biomarkers of disease progression, such as proteins and micro RNA molecules (miRNA). We tested and optimized a method for exosomal miRNA isolation from minimally invasive, small-volume serum samples obtained from white-tailed deer (Odocoileus virginianus). We confirmed the isolation of exosomes and optimized a commercially available benchtop kit to obtain sufficient and pure RNA for miRNA sequencing. The selected method for RNA extraction combines two 500-μL serum aliquots into one elution column and re-eluting the final product of the column. We identified 137 miRNA present in healthy white-tailed deer that can be used as a baseline to identify putative miRNA biomarkers of disease progression and mechanisms of infection in future comparative disease studies. This approach to biomarker discovery may help to inform biological processes in wildlife populations and provide alternatives to invasive or postmortem samples.

A dual-band microwave sensor for liquid permittivity characterization based on multiple complementary split-ring resonators

Abstract This article presents a dual-band planar microwave sensor to characterize the permittivity of liquid samples. The sensor utilizes a splitter–combiner microstrip segment loaded with two pairs of triangular-shaped complementary split-ring resonators (CSRRs). By integrating a meander slot into the CSRRs and incorporating inter-resonator coupling between the CSRRs, the proposed sensor achieves enhanced frequency shifts, resulting in improved sensitivity. An adulteration detection experiment is conducted to validate the sensor’s performance by mixing mineral oil into castor oil with a polydimethylsiloxane container placed on the sensing area. The variations in resonant frequency and peak attenuation are employed to extract the permittivity of the loaded liquid sample. The peak sensitivities in determining the real permittivity are measured to be 6.34% and 5.7% for the first and second frequency bands, respectively. The measured errors for extracting the real and imaginary parts of the complex permittivity are approximately 3.95% and 7.47% for the first frequency band and 3.67% and 6.28% for the second frequency band, respectively. The proposed dual-band microwave sensor, with its high sensitivity, compact size, small sample volume, and low cost, demonstrates great potential for applications in the quality monitoring of agricultural and industrial products.

Acoustotaxis‑based pump-less separation of highly motile human sperm by a SAW-in-capillary acoustofluidic platform

In artificial reproductive technologies mirroring in vivo fertilization, there is a correlation between sperm motility and fertilization outcomes. Here, we present a technology for separating sperm based on motility, which uses a biocompatible, pump-less, and simple acoustofluidic approach. Our developed and fabricated sperm separation configuration is an acoustofluidic platform consisting of a circular glass capillary tube mounted on top of a ZnO-based surface acoustic wave (SAW) device. It can separate highly motile sperm from small volumes of normal raw human semen samples. This configuration allows the generated traveling SAW to couple into the capillary, causing highly motile sperm cells to swim against the propagation direction of the traveling SAW along the capillary tube. We term this fascinating acoustic-induced sperm swimming behavior acoustotaxis in analogy with the well-known rheotaxis, in which sperm orientate to swim against a continuous fluid flow frequently generated by using an external pump in the microfluidic systems. Here, we benefit from the acoustotaxis for a pump-less separation of an effective highly motile sperm concentration from raw human semen samples at low volumes of up to 20–30 µL within a short period of about 4 minutes free from any centrifugation step. This sperm separation device exhibits a successful selection capability that leads to sperm progressive motility increase by 45 %, sperm curvilinear velocity by 23 %, sperm average path velocity by 69 %, and sperm linearity by 78 % in the selected cells in comparison with the raw sample. This acoustotaxic sperm selection separation approach provides an easy-to-use and effective method for the selection of highly motile sperm cells without adversely affecting sperm viability, paving the way for efficient and rapid sperm separation platforms, applicable for patients with low semen volumes, and up-scalable for a large volume of raw semen processing in a short time.

Gold-coated Impedance Biosensors on PCB and PET for Real-Time Monitoring of Cancer Cells

Abstract Electrical cell-substrate impedance sensing (ECIS) biosensors are widely used for in vitro cancer cell monitoring as they are label-free, require small sample volumes, and allow real-time monitoring. ECIS electrodes are typically made of pure gold, but the usage of pure gold electrodes is too costly for single-use applications. As an alternative, this work proposes the use of gold coatings on a printed sensor's electrodes. The interdigitated electrode design was used on glass fiber-reinforced epoxy resin for printed circuit boards (PCB), and polyethylene terephthalate (PET). The Cu/Ni electrodes on PCB were electroplated with Au, while the Cu/Ni electrodes on PET were coated with Au using an electroless technique. The physicochemical properties were studied using optical microscopy, scanning electron microscopy, and energy-dispersive spectroscopy. Electrochemical characterization was done using cyclic voltammetry and electrochemical impedance spectroscopy. Biocompatibility assessment and sensor functionality tests were done by culturing SiHa cervical cancer cell lines on these sensors and impedance measurements. The results show that both electroplated and electroless sensors were biocompatible and suitable to monitor SiHa cell growth. Electrochemical migration effect was observed on the sensors where the reaction occurred at 1.2V DC for the PCB sensor and 1.0V DC for the PET sensor.

Microfluidics in Assessing Platelet Function.

Microfluidics incorporate physiologically relevant substrates and flows that mimic the vasculature and are, therefore, a valuable tool for studying aspects of thrombosis and hemostasis. At high-shear environments simulating arterial flow, a microfluidic assay facilitates the study of platelet function, as platelet-rich thrombi form in a localized stenotic region of a flow channel. Utilizing devices that allow for small sample volume can additionally aid in evaluating platelet function under flow from volume-limited patient samples or animal models. Studying trauma patient samples or samples following platelet product transfusion may aid in directing therapeutic strategies for patient populations in which platelet function is critical. Effects of platelet inhibition via pharmacological agents can also be studied in this model. The objective of this protocol is to establish a microfluidic platform that incorporates physiologic flow, biological surfaces, and relevant hemostatic mechanisms to assess platelet function with implications for the study of trauma induced coagulopathy and transfusion medicine.

Surface-modified CMOS biosensors.

Biosensors translate biological events into electronic signals that quantify biological processes. They are increasingly used in in vitro diagnostics applications that leverage their ability to process small sample volumes. One recent trend has been to integrate biosensors with complementary metal-oxide-semiconductor (CMOS) chips to provide enhanced miniaturization, parallel sensing, and low power consumption at a low cost. CMOS-enabled biosensors are used in monitoring DNA hybridization, enzymatic reactions, and cell proliferation, to name a few applications. This paper explores the materials and processes used in emerging CMOS biosensors. We discuss subtractive and additive processes for creating electrodes for electrochemical sensing applications. We discuss functionalization techniques for creating bioelectronic interfaces that allow molecular events to be transduced into the electrical domain using a plurality of modalities that are readily provided by CMOS chips. Example modalities featured are optical sensing, electrochemical detection, electrical detection, magnetic sensing, and mechanical sensing.

Luminescent Terbium Probe for Time‐Resolved FRET and NSET Binding Assays with Quantum Dots and Gold Nanoparticles

AbstractTime‐gated or time‐resolved FRET (TR‐FRET) assays are important tools in biosensing, bioimaging, drug screening, and molecular diagnostics. Efficient TR‐FRET assays require stable lanthanide complexes with high absorption cross sections, high quantum yields, and long photoluminescence lifetimes. Owing to their challenging synthesis, such complexes are relatively rare and new components are of potential interest when developing TR‐FRET probes. Here, we evaluate the recently developed Tb complex CoraFluor‐1 concerning its analytical performance in terbium‐to‐quantum dot FRET and terbium‐to‐gold nanoparticle NSET assays using the prototypical biological recognition system of streptavidin and biotin. Biological binding was quantifiable at sub‐picomolar concentrations in small sample volumes, with broad applicability demonstrated across three commercial fluorescence plate readers used for time‐resolved, spectrally‐resolved, and clinical bioanalysis. Overall, CoraFluor‐1 provided excellent analytical performance as both FRET and NSET donor, validating its potential for developing new TR‐FRET probes for biosensing and bioimaging.

Roller compaction: Measuring ribbon porosity by terahertz spectroscopy and machine learning

Roller compaction is a crucial unit operation in pharmaceutical manufacturing, with its ribbon porosity widely recognised as a critical quality attribute. Terahertz spectroscopy has emerged as a fast and non-destructive technique to measure porosity in pharmaceutical products. From a sensing perspective, the irregular shape and uneven surface of fragmented ribbon pieces can affect the accuracy and precision of the measurements, particularly for techniques that probe only a small sampling volume. It is known that the porosity is not uniform within the ribbon structure, with variations expected across the width of the ribbon and in the microstructure corresponding to its surface texture. However, typical pharmaceutical analysis methods, such as envelope density, only report an average bulk porosity, are slow to operate and limited in accuracy. To address this challenge, we developed and trained convolutional neural network models using THz spectra as input to classify four types of topography typically encountered in ribbons: ridge, valley, flat plane and edge points. The classifiers achieved 91% validation accuracy in both identifying outliers and differentiating between ribbons of smooth and knurled surfaces. For the more challenging task of distinguishing between the ridges and valleys of knurled surfaces, an 81% testing accuracy was achieved. Once each measurement is paired with its topography, resolving the density distribution within the sample is possible. This data can be combined to arrive at an average bulk porosity value compatible with conventional pharmaceutical analysis.

Open-Channel Droplet Microfluidic Platform for Passive Generation of Human Sperm Microdroplets.

Sperm cryopreservation is important for many individuals across the globe. Recent studies show that vitrification is a valuable approach for maintaining sperm quality after freeze-thawing processes and requires sub-microliter to microliter volumes. A major challenge for the adoption of vitrification in fertility laboratories is the ability to pipette small volumes of sample. Here, we present an open droplet generator that leverages open-channel microfluidics to passively generate sub-microliter to microliter volumes of purified human sperm samples and preserves sperm kinematics. We conclude that our platform is compatible with human sperm, an important foundation for future implementation of vitrification in fertility laboratories.

Development of microflow ultra high performance liquid chromatography-mass spectrometry metabolomic assays for analysis of mammalian biofluids

Introduction and objectivesThe application of untargeted metabolomics assays using ultra high performance liquid chromatography-mass spectrometry (UHPLC-MS) to study metabolism in biological systems including humans is rapidly increasing. In some of these studies there is a requirement to collect and analyse low sample volumes of biofluids (e.g. tear fluid) or low cell and tissue mass samples (e.g. tissue needle biopsies). The application of microflow, capillary or nano liquid chromatography (≤ 1.0 mm column internal diameter (i.d.)) theoretically should accomplish a higher assay sensitivity compared to analytical liquid chromatography (2.1–5.0 mm column internal diameter). To date, there has been limited research into microflow UHPLC-MS assays that can be applied to study samples of low volume or mass.MethodsThis paper presents three complementary UHPLC-MS assays (aqueous C18 reversed-phase, lipidomics C18 reversed-phase and Hydrophilic Interaction Liquid Chromatography (HILIC)) applying 1.0 mm internal diameter columns for untargeted metabolomics. Human plasma and urine samples were applied for the method development, with porcine plasma, urine and tear fluid used for method assessment. Data were collected and compared for columns of the same length, stationary phase and stationary phase particle size but with two different column internal diameters (2.1 mm and 1.0 mm).Results and conclusionsAll three assays showed an increase in peak areas and peak widths when applying the 1.0 mm i.d. assays. HILIC assays provide an advantage at lower sample dilutions whereas for reversed phase (RP) assays there was no benefit added. This can be seen in the validation study where a much higher number of compounds were detected in the HILIC assay. RP assays were still appropriate for small volume samples with hundreds of compounds being detected. In summary, the 1.0 mm i.d. column assays are applicable for small volume samples where dilution is required during sample preparation.

Assay variables and early clinical evaluation of low-angle light scattering for platelet function analysis.

The recently developed platelet aggregation technique based on low-angle light scattering (LaSca) in diluted platelet-rich plasma (PRP) requires only a small sample volume and provides information about platelet aggregation and shape change. This study aimed to investigate the influence of preanalytical and analytical variables and to validate the method in a real-life pediatric hematology hospital setting. Platelet aggregation was induced by ADP in diluted PRP in the presence of 2mM calcium at 23°C. The study included healthy adults (n = 30), healthy children (n = 20), and pediatric patients with suspected or diagnosed platelet function abnormalities (n = 25). The assay parameters were stable for at least 3h after isolation of PRP and were sensitive to plasma dilution in the range of 2-8%. The initial aggregation velocity was significantly reduced in pediatric patients compared with healthy children (p < 0.05). ADP-induced light transmission amplitude was moderately correlated with LaSca amplitude of aggregation in healthy children (p = 0.52, p < 0.05) but not in pediatric patients. We standardized the protocol for platelet aggregation assessment by LaSca and characterized the influence of preanalytical and analytical variables on it.

Multimass Analysis of Adeno-Associated Virus Vectors by Orbitrap-Based Charge Detection Mass Spectrometry.

Adeno-associated virus (AAV) vectors have attracted significant attention as the main platform for gene therapy. To ensure the safety and efficacy of AAV vectors when used as gene therapy drugs, it is essential to assess their critical quality attributes (CQAs). These CQAs include the genome packaging status, the size of the genome encapsidated within the AAV capsid, and the stoichiometry of viral proteins (VPs) that constitute the AAV capsids. Analytical methods have been established for evaluating CQAs, such as analytical ultracentrifugation, capillary gel electrophoresis with laser-induced fluorescence detection, and capillary gel electrophoresis using sodium dodecyl sulfate with UV detection. Here, we present a multimass analysis of AAV vectors using orbitrap-based charge detection mass spectrometry (CDMS), a single-ion mass spectrometry. Orbitrap-based CDMS facilitates the quantitative evaluation of the genome packaging status based on the mass distribution of empty and full particles. Additionally, we established a novel method to analyze the encapsidated genome directly without pretreatment, such as protein digestion or heat treatment, and to estimate the stoichiometric variation of VP for the capsid based on the mass distribution constituted by the single peak corresponding to AAV particles. Orbitrap-based CDMS is a distinctive method that allows multiple mass characterizations of AAV vectors with a small sample volume of 20 μL for 1013 cp/mL in a short time (30 min), and it holds the potential to become a new standard method in the assessment of CQAs for AAV vectors.

Detection of renal tubular transporter proteins in canine urinary extracellular vesicles using liquid chromatography-tandem mass spectrometry.

Urinary extracellular vesicles (UEVs) are membranous particles that carry renal tubular transporter proteins. Here, we evaluate whether selected renal tubular transporter proteins can be detected in UEVs isolated from small volume (1-5 mL) canine urine samples of healthy dogs and canine patients with elevated circulating parathyroid hormone (PTH)/PTH-related peptide (PTHrp) concentrations, hypercortisolism, and primary hypoadrenocorticism using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The total creatinine content of each urine sample was calculated from urine volume and creatinine concentration. UEVs were isolated by size exclusion chromatography prior to quantification by nanoparticle tracking analysis and proteomic analysis by LC-MS/MS. Group comparisons were made using non-parametric statistics. Aquaporin-2 (AQP2) and the renal sodium/phosphate co-transporter (NPT2A) were detected in UEVs isolated from small volume samples of almost all healthy dogs but were not detected in most dogs with elevated circulating PTH/PTH related peptide (PTHrp) concentrations, hypercortisolism and primary hypoadrenocorticism. Total creatinine content of the urine sample was strongly positively correlated with the number of UEVs (rs = .84, P < .001); thus, total creatinine was used as a surrogate marker of UEV number. In healthy dogs, AQP2 and NPT2A were both detected in samples containing at least 1.7 × 109 UEVs or 24 μmol creatinine, however in non-healthy dogs, AQP2 and NPT2A were not detected in most samples containing up to 6.3 × 109 UEVs or 32 μmol creatinine.

In-Depth Chemical Analysis of the Brain Extracellular Space Using In Vivo Microdialysis with Liquid Chromatography-Tandem Mass Spectrometry.

Metabolomic analysis of samples acquired in vivo from the brain extracellular space by microdialysis sampling can provide insights into chemical underpinnings of a given brain state and how it changes over time. Small sample volumes and low physiological concentrations have limited the identification of compounds from this compartment, so at present, we have scant knowledge of its composition. As a result, most in vivo measurements have limited depth of analysis. Here, we describe an approach to (1) identify hundreds of compounds in brain dialysate and (2) routinely detect many of these compounds in 5 μL microdialysis samples to enable deep monitoring of brain chemistry in time-resolved studies. Dialysate samples collected over 12 h were concentrated 10-fold and then analyzed using liquid chromatography with iterative tandem mass spectrometry (LC-MS/MS). Using this approach on dialysate from the rat striatum with both reversed-phase and hydrophilic interaction liquid chromatography yielded 479 unique compound identifications. 60% of the identified compounds could be detected in 5 μL of dialysate without further concentration using a single 20 min LC-MS analysis, showing that once identified, most compounds can be detected using small sample volumes and shorter analysis times compatible with routine in vivo monitoring. To detect more neurochemicals, LC-MS analysis of dialysate derivatized with light and isotopically labeled benzoyl chloride was employed. 872 nondegenerate benzoylated features were detected with this approach, including most small-molecule neurotransmitters and several metabolites involved in dopamine metabolism. This strategy allows deeper annotation of the brain extracellular space than previously possible and provides a launching point for defining the chemistry underlying brain states.

Microfluidic-Integrated Chip Resonators for Electron Spin Sensing in Submicromolar, Submicroliter Solutions.

Planar or chip microresonators decrease the sample volume required for magnetic resonance spectroscopies to the nanoliter scale. However, the interrogation of nanoliter-scale solution samples on planar sensors is hindered by the lack of microfluidic devices that can simultaneously provide a small total volume and long-term sample stability. Here, we report microfluidic devices that decrease the total required sample volume to the submicroliter scale and also provide long-term physical stability and storability. We also report a 3D-printed microfluidic with a self-contained actuation mechanism, which allows the sample to be retracted from the microresonator surface for storage. The microfluidic devices are fabricated easily by laser cutting or 3D printing and are integrable with a broad range of planar sensors. We use planar inverse anapole (PIA) microresonators to obtain continuous wave (CW) electron paramagnetic resonance (EPR) spectra of natural-isotopic-abundance nitroxide radicals, which are ubiquitously used as reporters of biomolecular dynamics. We provide experimental evidence for a concentration sensitivity of 330 ± 40 nmol L-1, a concentration sensitivity limit of 800 ± 100 nmol L-1/mT√Hz, and an active volume no greater than 30 nL. Together, these developments represent an advance not only in the sensitivity of EPR spectroscopy but also in the design of microfluidics for stable, dead-volume-free placement of nanoliter-scale volumes of solutions on planar sensors.

Ultrasound Imaging of Nonlinear Media Response Using a Pressure-Dependent Nonlinearity Index

It has been shown that within the range of acoustic pressures used in ultrasound imaging, waveforms are distorted during propagation in tissue due to the physically nonlinear behavior of the tissue. This distortion leads to changes in the spectrum of the received ultrasound echoes, causing the transfer of signal energy from the fundamental frequency to higher harmonics. Interestingly, adipose tissue exhibits up to 50 % stronger nonlinear behavior compared to other soft tissues. The tissue nonlinearity parameter B/A is typically measured ex vivo using an ultrasound method in transmission mode, which requires extensive receiving systems. Currently, there is no improved ultrasound method for measuring the B/A nonlinearity parameter in vivo, which could be used in assessing the degree of fatty liver disease. We propose a new, simple approach to estimating nonlinear tissue properties. The proposed method involves transmitting ultrasound waves at significantly different acoustic pressures, recording echoes only in the fundamental frequency band at various depths, and introducing a nonlinearity index (NLI) based on specific echo amplitude ratios. The NLI at a given depth is calculated using the ratio of two dimensionless parameters. The first parameter is a predetermined constant obtained by dividing the total echo values from transmitting a signal at higher sound pressure by those from a signal at lower sound pressure, summed over a small tissue sample volume located near the transducer. The second parameter is calculated at a fixed distance from the transducer, determined by dividing the total echo values from transmitting a signal at higher sound pressure by those from a signal at lower pressure, summed over a small tissue volume of the tissue at that distance from the transducer. The reliability of the proposed measurements for assessing tissue nonlinearity has been substantiated through experimental confirmation of the existing correlations between the values of NLI and B/A in water, sunflower oil, and animal liver tissue samples with oil-enriched regions. The NLI was more than 15 % higher in sunflower oil than in water. The NLI in bovine liver sample below the area with injected oil (mimicking “steatosis”) was more than 35 % higher than in regions without oil. This method represents a promising modality for the nonlinear characterization of tissue regions in vivo, particularly for diagnosing fatty liver disease.