Real Biological Samples Research Articles

A disposable sensor based on one-pot synthesized tungsten oxide nanostructure-modified screen printed electrodes for selective detection of dopamine and uric acid.

Here, screen-printed carbon electrodes (SPCEs) were modified with ultrafine and mainly mono-disperse sea urchin-like tungsten oxide (SUWO3) nanostructures synthesized by a simple one-pot hydrothermal method for non-enzymatic detection of dopamine (DA) and uric acid (UA) in synthetic urine. Sea urchin-like nanostructures were clearly observed in scanning electron microscope images and WO3 composition was confirmed with XRD, Raman,FTIR and UV-Vis spectrophotometer. Modification of SPCEs with SUWO3 nanostructures via the drop-casting method clearly reduced the Rct value of the electrodes, lowered the ∆Ep and enhanced the DA oxidation current due to high electrocatalytic activity. As a result, SUWO3/SPCEs enabled highly sensitive non-enzymatic detection of DA (LOD: 51.4nM and sensitivity: 127µAmM-1cm-2) and UA (LOD: 253nM and sensitivity: 55.9µAmM-1cm-2) at low concentration. Lastly, SUWO3/SPCEs were tested with synthetic urine, in which acceptable recoveries for both molecules (94.02-105.8%) were obtained. Given the high selectivity, the sensor has the potential to be used for highly sensitive simultaneous detection of DA and UA in real biological samples.

Micromeria biflora mediated gold and silver nanoparticles for colourimetric detection of antibiotics and dyes degradation

The study reports on synthesis of aqueous extract mediated gold and silver nanoparticles of M. biflora (MBAuNPs and MBAgNPs) via hydrated chloroauric acid and silver nitrate salts. The nanoparticles (NPs) were produced in 1:15 (MBAuNPs) and 1:6 (MBAgNPs) ratios under sunlight displaying localized surface plasmon resonance (LSPR) peaks at 541 and 431 nm, respectively. The sizes characterized by transmission electron, scanning electron, and atomic force microscopic (SEM, TEM, AFM) techniques were respectively 26.73 nm and 53.81 nm. The subject NPs demonstrated application in the degradation of methylene blue, Congo red, Rhodamine B, methyl orange, ortho-nitrophenol, and para-nitrophenol ranging from 65 to 86 %. For detection of levofloxacin, amoxicillin, and azithromycin antibiotics, the MBAuNPs and MBAgNPs exhibited efficiency in real water and biological (blood plasma and urine) samples. Conclusively, the MBAuNPs and MBAgNPs applications for dyes degradation and antibiotics detection was found as simple and cost-effective analytical method.

Split aptazyme-based signal amplification for AβO analysis

Aptazyme is a chimera of functional nucleic acids, which integrates recognition and amplification elements to simplify the assay process and improve sensing efficiency. However, its application may be limited by signal leakage. In this work, we innovatively integrate the AβO aptamer and an MNAzyme (multicomponent nucleic acid enzyme) for highly efficient detection of AβO. The aptamer and half of the MNAzyme are positioned at one strand, and the other half of the MNAzyme is integrated with a toehold sequence. These two sequences cannot hybridize to activate the MNAzyme until the target is added. The background signal is significantly reduced by the split format and the secondary structure of DNA probes formed in the absence of the target. The proposed aptazyme can not only achieve amplification through enzymatic catalysis but also greatly improve the efficiency of signal transduction and output. We systematically investigated the influence of different DNA probes on the detection performance, and the optimized aptazyme can detect as low as 26.5 pM targets in 1h. The stability of this method was also investigated by detection targets in real biological samples.

Near-Infrared-Driven Nanorocket for Rapid and Ultrasensitive Detection of MicroRNA.

Herein, by introducing gold nanostars (AuNSs) as fuel core, a near-infrared-driven nanorocket (NIDNR) with pretty fast walking was exploited for ultrasensitive miRNA detection. Compared with traditional nanomaterials-comprised nanomachines (NMs), the NIDNR possesses much better kinetic and thermodynamic performance owing to the extra photothermal driving force from localized surface plasmon (LSP). Impressively, the whole reaction time of NIDNR down to 15 min was realized, which is almost more than 8 times beyond those of conventional DNA-based NMs. This way, the inherent obstacle of traditional NMs, including long reaction time and low efficiency, could be easily addressed. As a proof of concept, the NIDNR was successfully applied to develop an electrochemical biosensing platform for rapid and sensitive detection of miRNA with an LOD down to 2.95 aM and achieved the real-time assay of real biological samples from human hepatocellular carcinoma cells (MHCC97L) and HeLa, thus providing an innovative insight to design more versatile DNA nanomachines for ultimate application in biosensing platform construction and clinical sample detection.

Fluorescent immunochromatographic test strip for therapeutic drug monitoring of methotrexate with high sensitivity and wide dynamic range.

As a front-line chemotherapeutic drug for maintenance and consolidation therapy, methotrexate (MTX) has widely been applied to treat various tumors and some inflammatory diseases. However, because of its severe toxicity ascribed to low selectivity, it is necessary to monitor therapeutic drugs in high-dose MTX therapeutic regimens to ensure treatment safety. In this work, we developed a fluorescent immunochromatographic test strip (FITS) for monitoring MTX by employing time-resolved fluorescent microspheres as signal probes. With a competitive immunoassay mode, the FITS for MTX shows a super-wide dynamic range of 10pM-10μM, covering the entire clinical therapeutic concentration range of MTX. Therapeutic drug monitoring of MTX can be achieved within 7min with high specificity, facilitating the timely rescue of drug poisoning led by high-dose MTX treatment. The methodwasemployedfor monitoring MTX in the spiked human serum, urine, and milk, showing acceptable recoveriesranging from 94.0 to 110.0%. The established FITS has been applied toMTX detection in serum obtained from high-dose MTX treatment. The results from FITS and enzyme multiplied immunoassay technique showed no significant difference, suggesting its reliability for usage in real biological samples. The device shows promise in point-of-caretherapeutic drug monitoring for resource-limited countries and institutes, which significantly facilitates overcoming the lag time between sampling and results.

Graphene oxide-based colorimetric/fluorescence dual-mode immunochromatography assay for simultaneous ultrasensitive detection of respiratory virus and bacteria in complex samples

A point-of-care testing biosensor that supports direct, sensitive, and simultaneous identification of bacteria and virus is still lacking. In this study, an ultrasensitive immunochromatography assay (ICA) with colorimetric/fluorescence dual-signal output was proposed for flexible and accurate detection of respiratory virus and bacteria in complex samples. Colorimetric AuNPs of 16 nm and two layers of quantum dots (QDs) were coated onto the surface of monolayer graphene oxide (GO) layer by layer to form a multilayered dual-signal nanofilm. This material not only can generate strong colorimetric and fluorescence signals for ICA analysis but also can provide larger surface area, better stability, and superior dispersibility than conventional spherical nanomaterials. Two test lines were built onto the ICA strip to simultaneously detect common respiratory virus influenza A and respiratory bacteria Streptococcus pneumoniae. The dual-signal mode of assay greatly broadened the applied range of ICA method, in which the colorimetric mode allows for quick determination of virus/bacteria and the fluorescence mode ensures the highly sensitive and quantitative detection of target pathogens with detection limits down to 891 copies/mL and 17 cells/mL, respectively. The proposed dual-mode ICA can also be applied directly for real biological and environment samples, which suggests its great potential for field application.

Mass Spectrometry-Based Biosensing and Biopsy Technology

Sensitive and accurate detection of biomolecules by multiplexed methods is important for disease diagnosis, drug research, and biochemical analysis. Mass spectrometry has the advantages of high sensitivity, high throughput, and high resolution, making it ideal for biomolecular sensing. As a result of the development of atmospheric pressure mass spectrometry, researchers have been able to use a variety of means to identify target biomolecules and recognize the converted signals by mass spectrometry. In this review, three main approaches and tools are summarized for mass spectrometry sensing and biopsy techniques, including array biosensing, probe/pen-based mass spectrometry, and other biosensor–mass spectrometry coupling techniques. Portability and practicality of relevant mass spectrometry sensing methods are reviewed, together with possible future directions to promote the advancement of mass spectrometry for target identification of biomolecules and rapid detection of real biological samples.

Design of Polarity-Dependent Immunosensors Based on the Structural Analysis of Engineered Antibodies.

"Reagentless" immunosensors are emerging to address the challenge of practical and sensitive detection of important biomarkers in real biological samples without the need for multistep assays and user intervention, with applications ranging from research tools to point-of-care diagnostics. Selective target binding to an affinity reagent is detected and reported in one step without the need for washing or additional reporters. In this study, we used a structure-guided approach to identify a mutation site in an antibody fragment for the polarity-dependent fluorophore, Anap, such that upon binding of the protein target cardiac troponin I, the Anap-labeled antibody would produce a detectable and dose-dependent shift in emission wavelength. We observed a significant emission wavelength shift of the Anap-labeled anti-cTnI mutant, with a blue shift of up to 37 nm, upon binding to the cTnI protein. Key differences in the resulting emission spectra between target peptides in comparison to whole proteins were also found; however, the affinity and binding characteristics remained unaffected when compared to the wild-type antibody. We also highlighted the potential flexibility of the approach by incorporating a near-infrared dye, IRDye800CW, into the same mutation site, which also resulted in a dose-dependent wavelength shift upon target incubation. These reagents can be used in experiments and devices to create simpler and more efficient biosensors across a range of research, medical laboratory, and point-of-care platforms.

Electrochemical Monitoring of Sulfadiazine via La@CeO Incorporated with Reduced Graphene Oxide

In recent years, indiscriminate consumption and dumping of antibiotics have become destructive to human health and causes ecotoxicological pollution. Here, the irregular particle nanosized dendrite structure of lanthanum-doped cerium oxide (LCO) decorated with sheet-like reduced graphene oxide (RGO) composite was utilized to detect the sulfonamide-based drug sulfadiazine (SZ). LCO@RGO nanocomposite was prepared using the hydrothermal method, the synergistic effect between LCO and RGO facilitates electron transferability and conductivity which enhances the electrochemical properties toward the detection of SZ. The detection of SZ expressed a lower detection limit (0.005 µM) and linear range (0.01–265 µM) of the fabricated LCO@RGO/GCE electrode toward SZ, analyzed using the highly sensitive DPV technique. Also, DPV was utilized to determined shows good repeatability, reproducibility, and storage stability of fabricated LCO@RGO/GCE. Moreover, effective practicability was proven in human blood serum and river water samples with great recovery results. All the above probes the synthesized LCO@RGO’s thriving and outstanding electrocatalytic performance of this nanocomposite’s highly sensitive detection of SZ in real biological and environmental samples.

Construction of a novel sensor based on activated nanodiamonds, zinc oxide, and silver nanoparticles for the determination of a selective inhibitor of cyclic guanosine monophosphate in real biological and food samples

In the present work, we developed a highly sensitive and selective method for the determination of sildenafil (SIL) using a modified glassy carbon electrode (GCE) with activated nanodiamonds, zinc oxide, and silver nanoparticles nanocomposite (AND@ZnO-Ag/GCE). Various voltammetric techniques, including CV, DPV, and EIS, were employed to investigate the electrochemical process of SIL on the modified electrode. The resulting AND@ZnO-Ag/GCE electrode exhibited enhanced electron transfer between the electrode and the analyte, resulting in an excellent electrocatalytic performance for SIL oxidation under optimized conditions. The analytical performance of the AND@ZnO-Ag/GCE sensor demonstrated two linear responses within the concentration range of 0.01–0.08 μM and 0.08–14.6 μM, with a remarkably low detection limit of 7.08 nM. Moreover, the modified sensor (AND@ZnO-Ag/GCE) exhibited high selectivity and anti-interference capacity. It successfully detected trace amounts of SIL in biological and food samples, yielding satisfactory results. Furthermore, this work provides insights into the potential adulteration of energy drinks with sildenafil, addressing an area that has received limited attention in previous research.

Quantification of tramadol and serotonin by cobalt nickel tungstate in real biological samples to evaluate the effect of analgesic drugs on neurotransmitters

In this work, CoNiWO4 nanocomposite was used as an electrochemical sensor for the simultaneous electrochemical detection of tramadol and serotonin. The nanocomposite was synthesized using a hydrothermal method and characterized via XRD, SEM, TGA, Zeta, UV, and FTIR. The sensor was developed by depositing CoNiWO4-NPs onto the glassy carbon electrode surface. Tramadol and serotonin were detected by employing cyclic voltammetry (CV), differential pulse voltammetry (DPV), electrochemical impedance spectroscopy (EIS), and chronoamperometry. Analytes were detected at different pH, concentrations, and scan rates. The prepared sensor showed a 0–60 µM linear range, with a LOD of 0.71 µM and 4.29 µM and LOQ of 14.3 µM and 2.3 µM for serotonin and tramadol, respectively. Finally, the modified electrode (CoNiWO4–GCE) was applied to determine tramadol and serotonin in biological samples.

A sensitive “turn-off-on” fluorescence platform for bilirubin detection based on conjugated polymer nanoparticles and ferric ions

Bilirubin is an important biomarker of jaundice, and the quantitative detection of bilirubin is of great significance in the diagnosis and prevention of early jaundice. In this paper, Fe3+ quenched conjugated polymer nanoparticles were used as a "turn-off-on" fluorescence platform to detect bilirubin for the first time. The CPNsPBMC-COOH were prepared through a nanoprecipitation method based on polystyrene maleic anhydride and PBMC, a conjugated polymer synthesized by our research group. The fluorescence of CPNsPBMC-COOH can be efficiently quenched closely to 95% by Fe3+. The optimized fluorescence platform was obtained (CPNsPBMC-COOH: 10 μg/mL, Fe3+: 4.5 μM). The platform to detect bilirubin has a higher sensitivity with a 5 nM of LOD. The detection range was in the 1.32–26.68 μM, which can cover the concentration of bilirubin in normal adult serum (<25 μM). Moreover, the stoichiometries and binding constants of CPNsPBMC-COOH, bilirubin, and Fe3+ are obtained. The binding ability is better for bilirubin and Fe3+. The static quenching mode is found by the absorption spectra and the fluorescence lifetimes. Lastly, this method has obtained satisfactory results in real biological samples (mouse serum and mouse plasma). This work can be used for daily rapid clinical bilirubin detection.

A Schiff base fluorescence switch-on probe derived from vitamin B6 cofactor for simultaneous detection of bovine serum albumin and ovalbumin

The abnormal amount of albumin found in serum and urine samples is an important biomarker for a variety of illnesses. It is essential for clinical diagnosis, medicinal research, and preventative medicine to correctly assess the albumin concentration in actual biological samples. Herein, vitamin B6 cofactors derived Schiff bases obtained by condensing pyridoxal 5′-phosphate (PLP) with 2-aminophenol (L), pyridoxal with 2-aminophenol (L1) and PLP with aniline (L2) were employed for the detection of albumins. Schiff base L is non-fluorescent due to conformational flexibility and free intramolecular rotation, but becomes fluorescent upon protein–ligand complex formation with bovine serum albumin (BSA) and ovalbumin (OVA). Other Schiff bases L1 and L2 failed to response the presence of BSA and OVA. The intensity of fluorescence of L increases linearly with the increasing concentration of BSA and OVA, and the detection limits for BSA and OVA were estimated to be 0.18 µM and 0.13 µM, respectively. The molecular docking and molecular dynamics simulations were done to investigate the binding affinity and modes between the albumins (BSA and OVA) and L. The developed detection method was applied for the detection of BSA and OVA in real biological samples of milk, serum, egg white and urine, with satisfactory recovery.

Ratiometric optical detection of pyrophosphate based on aggregation-caused dual-signal response of gold nanoclusters.

Sensing of pyrophosphate ion (PPi) has received much attention due to the strong demand for clinical diagnostics. Herein, based on gold nanoclusters (Au NCs), a ratiometric optical detection method for PPi is developed by simultaneously detecting the dual-signals of fluorescence (FL) and second-order scattering (SOS). The PPi is detected by inhibiting the formation of aggregates of Fe3+ with Au NCs. Binding of Fe3+ to Au NCs causes aggregation of Au NCs, which leads to fluorescence quenching and scattering increasing. The presence of PPi can competitively bind Fe3+ to re-disperse the Au NCs and finally recover the fluorescence and reduce the scattering signal. The designed PPi sensor shows a high sensitivity with a linear range of 5-50 μM and a detection limit of 1.2 μM. In addition, the assay has excellent selectivity for PPi, which makes its application in real biological samples extremely valuable.

Evaluation of DDA Library-Free Strategies for Phosphoproteomics and Ubiquitinomics Data-Independent Acquisition Data.

Phosphoproteomics and ubiquitinomics data-independent acquisition (DIA) mass spectrometry (MS) data is typically analyzed by using a data-dependent acquisition (DDA) spectral library. The performance of various library-free strategies for analyzing phosphoproteomics and ubiquitinomics DIA MS data has not been evaluated. In this study, we systematically compare four commonly used DDA library-free approaches including Spectronaut's directDIA, DIA-Umpire, DIA-MSFragger, and in silico-predicted library for analysis of phosphoproteomics SWATH, DIA, and diaPASEF data as well as ubiquitinomics diaPASEF data. Spectronaut's directDIA shows the highest sensitivity for phosphopeptide detection not only in synthetic phosphopeptide samples but also in phosphoproteomics SWATH-MS and DIA data from real biological samples, when compared to the other three library-free strategies. For phosphoproteomics diaPASEF data, Spectronaut's directDIA and the in silico-predicted library based on DIA-NN identify almost the same number of phosphopeptides as a project-specific DDA spectral library. However, only about 30% of the total phosphopeptides are commonly identified, suggesting that the library-free strategies for phospho-diaPASEF data need further improvement in terms of sensitivity. For ubiquitinomics diaPASEF data, the in silico-predicted library performs the best among the four workflows and detects ∼50% more K-GG peptides than a project-specific DDA spectral library. Our results demonstrate that Spectronaut's directDIA is suitable for the analysis of phosphoproteomics SWATH-MS and DIA MS data, while the in silico-predicted library based on DIA-NN shows substantial advantages for ubiquitinomics diaPASEF MS data.

Near-infrared magnetic core-shell nanoparticles based on lanthanide metal-organic frameworks as a ratiometric felodipine sensing platform

Felodipine is an effective drug to treat hypertension, but its abuse can cause bardycardia. It is significant to develop highly sensitive detection platform for felodipine to enable the efficient treatment of hypertension diseases. In this work, to highly efficiently detect felodipine, multi-emission near-infrared (NIR) hierarchical magnetic core-shell lanthanide-MOF nanoparticles, namely Nd-MOF@Yb-MOF@SiO2@Fe3O4 (NIR-1), has been synthesized by layer-by-layer (LBL) method. LBL method can adjust the optical properties of NIR-1 and expose more active sites to improve sensitivity in detection process. NIR-1 has near-infrared luminescence emission, which can efficiently avoid the interference of autofluorescence in biological tissues. Photo-luminescent (PL) experiments also reveal that NIR-1 could be used as a near-infrared ratiometric luminescent sensor for felodipine detection with high selectivity and sensitivity, the low of detection limit (LOD) is 6.39 nM in felodipine detection, which is also performed using real biological samples. In addition, NIR-1 can be used as a ratiometric thermometer could also be applied in the temperature sensing from 293 K to 343 K. Finally, detection mechanisms for felodipine and temperature sensing performance based on near-infrared (NIR) emission were also investigated and discussed in detail.

Hybrid Nanomaterial of Graphene Oxide Quantum Dots with Multi-Walled Carbon Nanotubes for Simultaneous Voltammetric Determination of Four DNA Bases.

In this proof-of-concept study, a novel hybrid nanomaterial-based electrochemical sensor was developed for the simultaneous detection of four DNA bases. For the modification of the working electrode surface, graphene oxide quantum dots (GOQDs) were synthesized using a solvothermal method. GOQDs were then used for the preparation of a hybrid nanomaterial with multi-walled carbon nanotubes (GOQD-MWCNT) using a solvothermal technique for the first time. Transmission electron microscopy (TEM) was used to characterize the GOQDs-MWCNTs. A glassy carbon electrode (GCE) was modified with the GOQDs-MWCNTs using Nafion™ to prepare a GOQD-MWCNT/GCE for the simultaneous determination of four DNA bases in phosphate buffer solution (PBS, pH 7.0) using differential pulse voltammetry (DPV). The calibration plots were linear up to 50, 50, 500, and 500 µM with a limit of detection at 0.44, 0.2, 1.6, and 5.6 µM for guanine (G), adenine (A), thymine (T) and cytosine (C), respectively. The hybrid-modified sensor was used for the determination of G, A, T, and C spiked in the artificial saliva samples with the recovery values ranging from 95.9 to 106.8%. This novel hybrid-modified electrochemical sensor provides a promising platform for the future development of a device for cost-effective and efficient simultaneous detection of DNA bases in real biological and environmental samples.

Catalytic Modification of Porous Two-Dimensional Ni-MOFs on Portable Electrochemical Paper-Based Sensors for Glucose and Hydrogen Peroxide Detection.

Rapid and accurate detection of changes in glucose (Glu) and hydrogen peroxide (H2O2) concentrations is essential for the predictive diagnosis of diseases. Electrochemical biosensors exhibiting high sensitivity, reliable selectivity, and rapid response provide an advantageous and promising solution. A porous two-dimensional conductive metal-organic framework (cMOF), Ni-HHTP (HHTP = 2,3,6,7,10,11-hexahydroxytriphenylene), was prepared by using a one-pot method. Subsequently, it was employed to construct enzyme-free paper-based electrochemical sensors by applying mass-producing screen-printing and inkjet-printing techniques. These sensors effectively determined Glu and H2O2 concentrations, achieving low limits of detection of 1.30 μM and 2.13 μM, and high sensitivities of 5573.21 μA μM-1 cm-2 and 179.85 μA μM-1 cm-2, respectively. More importantly, the Ni-HHTP-based electrochemical sensors showed an ability to analyze real biological samples by successfully distinguishing human serum from artificial sweat samples. This work provides a new perspective for the use of cMOFs in the field of enzyme-free electrochemical sensing, highlighting their potential for future applications in the design and development of new multifunctional and high-performance flexible electronic sensors.

Recognition Receptor for Methylated Arginine at the Single Molecular Level.

Among the various types of post-translational modifications (PTMs), methylation is the simple functionalized one that regulates the functions of proteins and affects interactions of protein-protein and protein-DNA/RNA, which will further influence diverse cellular processes. The methylation modification has only a slight effect on the size and hydrophobicity of proteins or peptides, and it cannot change their net charges at all, so the methods for recognizing methylated protein are still limited. Here, we designed a recognition receptor consisting of a α-hemolysin (α-HL) nanopore and polyamine decorated γ-cyclodextrin (am8γ-CD) to differentiate the methylation of peptide derived from a heterogeneous nuclear ribonucleoprotein at the single molecule level. The results indicate that the modification of a methyl group enhances the interaction between the peptide and the recognition receptor. The results of molecular simulations were consistent with the experiments; the methylated peptide interacts with the receptor strongly due to the more formation of hydrogen bonds. This proposed strategy also can be used to detect PTM in real biological samples and possesses the advantages of low-cost and high sensitivity and is label-free. Furthermore, the success in the construction of this recognition receptor will greatly facilitate the investigation of pathogenesis related to methylated arginine.

Rapid Detection of Ag(I) via Size-Induced Photoluminescence Quenching of Biocompatible Green-Emitting, l-Tryptophan-Scaffolded Copper Nanoclusters.

Atomically precise metal nanoclusters capped with small molecules like amino acids are highly favored due to their specific interactions and easy incorporation into biological systems. However, they are rarely explored due to the challenge of surface functionalization of nanoclusters with small molecules. Herein, we report the synthesis of a green-emitting (λex = 380 nm, λem = 500 nm), single-amino-acid (l-tryptophan)-scaffolded copper nanocluster (Trp-Cu NC) via a one-pot route under mild reaction conditions. The synthesized nanocluster can be used for the rapid detection of a heavy metal, silver (Ag(I)), in the nanomolar concentration range in real environmental and biological samples. The strong green photoluminescence intensity of the nanocluster quenched significantly upon the addition of Ag(I) due to the formation of bigger nanoparticles, thereby losing its energy quantization. A notable color change from light yellow to reddish-brown can also be observed in the presence of Ag(I), allowing its visual colorimetric detection. Portable paper strips fabricated with the Trp-Cu NC can be reliably used for on-site visual detection of Ag(I) in the micromolar concentration range. The Trp-Cu NC possesses excellent biocompatibility, making it a suitable nanoprobe for cell imaging; thus, it can act as an in vivo biomarker. The nanocluster showed a significant spectral overlap with anticancer drug doxorubicin and thus can be used as an effective fluorescence resonance energy transfer (FRET) pair. FRET results can reveal important information regarding the attachment of the drug to the nanocluster and hence its role as a potential drug carrier for targeted drug delivery within the human body.