Nonspecific Aggregation Research Articles

Selective-Integrative Technology for the Separation of Colostrum Into Components and the Possibilities of Obtaining Protein Substances From Different Sources

Background. Obtaining biologically active natural compounds involved in the regulation of metabolism is an important goal in biotechnology. Colostrum is a unique natural source of various biologically active compounds. However, the extremely high natural variability of colostrum composition does not meet the existing requirements for standardization in pharmaceutical preparations. Objective. To develop a method for separating colostrum into its basic components (lipids, casein, and protein fractions), thereby reducing the variability of whole colostrum composition, obtaining several target products, and demonstrating the possibility of acquiring new protein substances from different sources. Methods. Colostrum separation was carried out through centrifugation and membrane filtration. Plant proteins (sunflower) and milk proteins were used to obtain protein substances from different sources. The composition of proteins, carbohydrates, and nucleic acids was determined using mass spectrometry, centrifugation, and membrane filtration. Results. The proposed method for obtaining basic substances from colostrum significantly reduced the vari­ability in composition compared to whole colostrum. The efficiency of protein sedimentation in concentrated protein solutions by centrifugation and ultrafiltration was shown to depend on protein concentration. Additionally, the formation of non-specific protein aggregates in the centrifugal field allowed the extraction of protein substances from various natural sources, which is relevant for functional nutrition. Conclusions. The proposed selective-integrative technology for obtaining different substances from colostrum significantly reduces the high variability of whole colostrum composition. It increases the efficiency of component separation into lipid, casein fractions, low molecular weight protein fractions, and ultrafiltrate, while also enabling the acquisition of protein substances from diverse sources.

Lipoic acid-locked reduction-responsive core-cross-linked micelles delivering paclitaxel for triple-negative breast cancer treatment

Most anti-tumor agents suffer from systemic non-specific distribution and low aggregation in tumors, which not only decreases the therapeutic efficacy, but also causes systemic toxic side effects in the treatments of tumors. In recent years, the rapid development of nanotechnology has brought new ideas for the application of anti-tumor drugs. Nanomedicines, such as liposomes and micelles, can improve drug targeting and prolong systemic circulation time to promote anti-tumor efficacy and reduce toxic side effects. However, conventional micelles bear the risk of instability and premature drug leaking in the blood circulation. We designed a reduction-responsive core-cross-linked micelle PTX@Fmoc-LA-PEG efficiently encapsulating Paclitaxel (PTX) via π-π stacking and hydrophobic interactions of Fmoc and PTX. Moreover, the micelle was further locked based on the cross-linking properties of the disulfide bonds formed by lipoic acid (LA). As expected, the core-cross-linked micelles PTX@Fmoc-LA-PEG remained stable in normal physiological environments, while restoring the normal drug release rate of micelles under the highly reducing environment due to LA unlocking. The blank micelles (Fmoc-LA-PEG) exhibited excellent biocompatibility, while the drug-loaded micelles (PTX@Fmoc-LA-PEG) displayed a remarkable anti-tumor effect in vitro and in vivo experiments. These results suggested that core-cross-linked micelles PTX@Fmoc-LA-PEG have great potential to improve the targeting and stability of anti-tumor drugs.

Application of Machine Learning in the Quantitative Analysis of the Surface Characteristics of Highly Abundant Cytoplasmic Proteins: Toward AI-Based Biomimetics.

Proteins in the crowded environment of human cells have often been studied regarding nonspecific interactions, misfolding, and aggregation, which may cause cellular malfunction and disease. Specifically, proteins with high abundance are more susceptible to these issues due to the law of mass action. Therefore, the surfaces of highly abundant cytoplasmic (HAC) proteins directly exposed to the environment can exhibit specific physicochemical, structural, and geometrical characteristics that reduce nonspecific interactions and adapt to the environment. However, the quantitative relationships between the overall surface descriptors still need clarification. Here, we used machine learning to identify HAC proteins using hydrophobicity, charge, roughness, secondary structures, and B-factor from the protein surfaces and quantified the contribution of each descriptor. First, several supervised learning algorithms were compared to solve binary classification problems for the surfaces of HAC and extracellular proteins. Then, logistic regression was used for the feature importance analysis of descriptors considering model performance (80.2% accuracy and 87.6% AUC) and interpretability. The HAC proteins showed positive correlations with negatively and positively charged areas but negative correlations with hydrophobicity, the B-factor, the proportion of beta structures, roughness, and the proportion of disordered regions. Finally, the details of each descriptor could be explained concerning adaptative surface strategies of HAC proteins to regulate nonspecific interactions, protein folding, flexibility, stability, and adsorption. This study presented a novel approach using various surface descriptors to identify HAC proteins and provided quantitative design rules for the surfaces well-suited to human cellular crowded environments.

Effects of environmental temperature during maturation on protein characteristics in spring wheat (Triticum aestivum cv. Haruyokoi)

Wheat (Triticum aestivum cv. Haruyokoi) gluten proteins are important contributors to wheat flour quality. Numerous studies have examined the effects of heat stress on gluten proteins. We clarified the relationship between growth temperature and the characteristics of storage proteins in spring wheat grown under mild conditions (<30 °C). As the growth temperature increased, crude protein content also increased and the activity of protein disulfide isomerase (PDI), which is involved in the folding and transport of gluten proteins, decreased. The growth temperature also affected the amount of aggregated storage proteins, with an increase in sodium dodecyl sulfate (SDS)-insoluble proteins observed under high-temperature conditions. From these results, it was inferred that high growth temperatures caused an environment where nonspecific aggregation is likely to occur. The accumulation of specific proteins was also affected. The high-molecular-weight glutenin subunit (HMW-GS) Dx5 encoded by Glu-D1d allele, which has a marked impact on the viscoelastic properties of dough characteristics, showed high accumulation under high growth temperatures. These effects of growth temperature on storage proteins appear to affect the formation of glutenin macropolymer, which in turn affects the physical properties of the dough. As global warming continues, this study could provide helpful information for long-term predictions of wheat quality.

Real-Time Monitoring of Multitarget Antimicrobial Mechanisms of Peptoids Using Label-Free Imaging with Optical Diffraction Tomography.

Antimicrobial peptides (AMPs) are promising therapeutics in the fight against multidrug-resistant bacteria. As a mimic of AMPs, peptoids with N-substituted glycine backbone have been utilized for antimicrobials with resistance against proteolytic degradation. Antimicrobial peptoids are known to kill bacteria by membrane disruption; however, the nonspecific aggregation of intracellular contents is also suggested as an important bactericidal mechanism. Here,structure-activity relationship (SAR) of a library of indole side chain-containing peptoids resulting in peptoid 29 as a hit compound is investigated. Then, quantitative morphological analyses of live bacteria treated with AMPs and peptoid 29 in a label-free manner using optical diffraction tomography (ODT) are performed. It is unambiguously demonstrated that both membrane disruption and intracellular biomass flocculation are primary mechanisms of bacterial killing by monitoring real-time morphological changes of bacteria. These multitarget mechanisms and rapid action can be a merit for the discovery of a resistance-breaking novel antibiotic drug.

Real-time monitoring of large-scale crystal growth using batch crystallization for serial crystallography

Serial crystallography (SX) enable to visualize the molecular dynamics of macromolecule at room temperature with minimal radiation damage. Large-scale batch crystallization method is widely applied for the SX sample preparation, but the simultaneous growth of multiple crystals in the batch is not well-studied. In this study, we report the real-time monitoring of large-scale crystal growth during batch crystallization using the protein lysozyme. Highly concentrated lysozyme immediately formed random-sized aggregates upon mixing with the crystallization solution. The nucleation and crystal growth started from the aggregation area. In regions with high aggregation density, the size of initially grown crystals increased rather than further nucleation and crystal formation. The qualities of the crystals obtained from the large-scale batch crystallization method were verified by serial synchrotron crystallography, which determined the room-temperature structure of lysozyme at 1.6 Å. Based on the activation energy and nucleation barrier, our results indicate that a high concentration of lysozyme initially forms non-specific aggregates, a process that involves nucleation from non-specific aggregates. Then, the nonspecifically aggregated proteins nucleate to form crystals that grow into larger crystals. The findings of this study provide a better understanding of the phenomenon of a large number of crystal growths in large-scale batch crystallization.

Profiling the Hsp70 Chaperone Network in Heat-Induced Proteotoxic Stress Models of Human Neurons.

Heat stroke is among the most hazardous hyperthermia-related illnesses and an emerging threat to humans from climate change. Acute brain injury and long-lasting brain damage are the hallmarks of this condition. Hyperthermic neurological manifestations are remarkable for their damage correlation with stress amplitude and long-term persistence. Hyperthermia-induced protein unfolding, and nonspecific aggregation accumulation have neurotoxic effects and contribute to the pathogenesis of brain damage in heat stroke. Therefore, we generated heat-induced, dose-responsive extreme and mild proteotoxic stress models in medulloblastoma [Daoy] and neuroblastoma [SH-SY5Y] and differentiated SH-SY5Y neuronal cells. We show that heat-induced protein aggregation is associated with reduced cell proliferation and viability. Higher protein aggregation in differentiated neurons than in neuroblastoma precursors suggests a differential neuronal vulnerability to heat. We characterized the neuronal heat shock response through RT-PCR array analysis of eighty-four genes involved in protein folding and protein quality control (PQC). We identify seventeen significantly expressed genes, five of which are Hsp70 chaperones, and four of their known complementing function proteins. Protein expression analysis determined the individual differential contribution of the five Hsp70 chaperones to the proteotoxic stress response and the significance of only two members under mild conditions. The co-expression analysis reveals significantly high co-expression between the Hsp70 chaperones and their interacting partners. The findings of this study lend support to the hypothesis that hyperthermia-induced proteotoxicity may underlie the brain injury of heat stroke. Additionally, this study presents a comprehensive map of the Hsp70 network in these models with potential clinical and translational implications.

Nonspecific Amyloid Aggregation of Chicken Smooth-Muscle Titin: In Vitro Investigations.

A giant multidomain protein of striated and smooth vertebrate muscles, titin, consists of tandems of immunoglobulin (Ig)- and fibronectin type III (FnIII)-like domains representing β-sandwiches, as well as of disordered segments. Chicken smooth muscles express several titin isoforms of ~500-1500 kDa. Using various structural-analysis methods, we investigated in vitro nonspecific amyloid aggregation of the high-molecular-weight isoform of chicken smooth-muscle titin (SMTHMW, ~1500 kDa). As confirmed by X-ray diffraction analysis, under near-physiological conditions, the protein formed amorphous amyloid aggregates with a quaternary cross-β structure within a relatively short time (~60 min). As shown by circular dichroism and Fourier-transform infrared spectroscopy, the quaternary cross-β structure-unlike other amyloidogenic proteins-formed without changes in the SMTHMW secondary structure. SMTHMW aggregates partially disaggregated upon increasing the ionic strength above the physiological level. Based on the data obtained, it is not the complete protein but its particular domains/segments that are likely involved in the formation of intermolecular interactions during SMTHMW amyloid aggregation. The discovered properties of titin position this protein as an object of interest for studying amyloid aggregation in vitro and expanding our views of the fundamentals of amyloidogenesis.

Water-Soluble and Degradable Gelatin/Polyaniline Assemblies with a High Photothermal Conversion Efficiency for pH-Switchable Precise Photothermal Therapy.

Photothermal therapy (PTT) is regarded as one of the potential techniques to replace surgery in the treatment of tumors. Polyaniline (PANI) shows better biocompatibility than inorganic reagents, which has been widely used in tumor photoacoustic (PA) imaging and PTT. However, the poor water solubility and nonspecific aggregation of PANI nanoparticles severely restricted their biomedical application. In addition, it is difficult to control the photothermal effect just on cancer cells. Herein, we develop tumor pH-responsive PANI-Gel/Cu assemblies, which can achieve targeted and precise ablation of tumors. Due to the high hydrophilicity of gelatin, the PANI-Gel/Cu assemblies show excellent dispersion in physiological solutions and long-term stability. By taking advantage of the self-doping effect between the carboxyl groups in gelatin and the imine part of the PANI skeleton, the photothermal characteristics of PANI-Gel/Cu assemblies can be promoted effectively by the acid tumor microenvironment, and the PA imaging of PANI-Gel/Cu assemblies can also be activated by tumor pH. Consequently, both the PTT enhancement and PA signal amplification can be triggered under a tumor microenvironment, and PANI-Gel/Cu assemblies can be targeted to cancer cells with the RGD sequences in their gelatin skeleton. In vivo imaging-guided PTT to A549 cancer shows precise treatment with little harm to normal cells, and PANI-Gel/Cu assemblies can disassemble into tiny particles (<15 nm) under laser irradiation. This work overcomes the intrinsic limitation of PANI materials, i.e., poor water solubility and nonspecific aggregation, meanwhile providing a pH-active PANI-based platform for precise and effective ablation of cancer.

Updates in idiopathic pulmonary hemosiderosis in 2022: A state of the art review.

This manuscript reports the recent advances in idiopathic pulmonary hemosiderosis (IPH), a rare cause of diffuse alveolar hemorrhagein children and adults. This narrative review of the literature summarizes different aspects of IPH, including proposed pathogenesis, patient demographics, clinicaland radiological characteristics, treatment, and prognosis. Additionally, the association between Celiac Disease (CD) and IPH is carefully evaluated. IPH is a frequently misdiagnosed disease. The delay in the diagnosis of IPH is often significant but fortunately, appears to have decreased in recent years. IPH in adults and children have distinct demographic preferences. The autoantibodies are common in IPH but with a definite difference between the adult and pediatric populations. The definitive diagnosis of IPH requires lung biopsy and careful exclusion of all competing diagnoses, even with lung biopsy showing bland pulmonary hemorrhage. The presence of nonspecific inflammatory cells or lymphoid aggregates may suggest a secondary immunologic phenomenon and needs careful evaluation and follow-up. A substantial number of patients suffer from coexisting CD, also known as Lane-Hamilton syndrome (LHS), and all patients with IPH need to be evaluated for LHS by serology. Although strict gluten free dietcan manage the majority of patients with LHS, other patients generally require immunosuppressive therapy. The corticosteroidsare the backbone of IPH therapy. Recently utilized experimental treatment options include mesenchymal stem cell transplant, liposteroid and bronchial artery embolization. The immunosuppression should be adjusted to achieve optimal disease control. Patients may progress to end-stage lung disease despite all measures, and lung transplantation may be the only viable option.

Human testis–specific Y-encoded protein-like protein 5 is a histone H3/H4-specific chaperone that facilitates histone deposition in vitro

DNA and core histones are hierarchically packaged into a complex organization called chromatin. The nucleosome assembly protein (NAP) family of histone chaperones is involved in the deposition of histone complexes H2A/H2B and H3/H4 onto DNA and prevents nonspecific aggregation of histones. Testis-specific Y-encoded protein (TSPY)–like protein 5 (TSPYL5) is a member of the TSPY-like protein family, which has been previously reported to interact with ubiquitin-specific protease USP7 and regulate cell proliferation and is thus implicated in various cancers, but its interaction with chromatin has not been investigated. In this study, we characterized the chromatin association of TSPYL5 and found that it preferentially binds histone H3/H4 via its C-terminal NAP-like domain both in vitro and ex vivo. We identified the critical residues involved in the TSPYL5–H3/H4 interaction and further quantified the binding affinity of TSPYL5 toward H3/H4 using biolayer interferometry. We then determined the binding stoichiometry of the TSPYL5–H3/H4 complex in vitro using a chemical cross-linking assay and size-exclusion chromatography coupled with multiangle laser light scattering. Our results indicate that a TSPYL5 dimer binds to either two histone H3/H4 dimers or a single tetramer. We further demonstrated that TSPYL5 has a specific affinity toward longer DNA fragments and that the same histone-binding residues are also critically involved in its DNA binding. Finally, employing histone deposition and supercoiling assays, we confirmed that TSPYL5 is a histone chaperone responsible for histone H3/H4 deposition and nucleosome assembly. We conclude that TSPYL5 is likely a new member of the NAP histone chaperone family.

A second chance for protein targeting/folding: Ubiquitination and deubiquitination of nascent proteins.

Molecular chaperones in cells constantly monitor and bind to exposed hydrophobicity in newly synthesized proteins and assist them in folding or targeting to cellular membranes for insertion. However, proteins can be misfolded or mistargeted, which often causes hydrophobic amino acids to be exposed to the aqueous cytosol. Again, chaperones recognize exposed hydrophobicity in these proteins to prevent nonspecific interactions and aggregation, which are harmful to cells. The chaperone-bound misfolded proteins are then decorated with ubiquitin chains denoting them for proteasomal degradation. It remains enigmatic how molecular chaperones can mediate both maturation of nascent proteins and ubiquitination of misfolded proteins solely based on their exposed hydrophobic signals. In this review, we propose a dynamic ubiquitination and deubiquitination model in which ubiquitination of newly synthesized proteins serves as a "fix me" signal for either refolding of soluble proteins or retargeting of membrane proteins with the help of chaperones and deubiquitinases. Such a model would provide additional time for aberrant nascent proteins to fold or route for membrane insertion, thus avoiding excessive protein degradation and saving cellular energy spent on protein synthesis. Also see the video abstract here: https://youtu.be/gkElfmqaKG4.

De novo design and directed folding of disulfide-bridged peptide heterodimers

Peptide heterodimers are prevalent in nature, which are not only functional macromolecules but molecular tools for chemical and synthetic biology. Computational methods have also been developed to design heterodimers of advanced functions. However, these peptide heterodimers are usually formed through noncovalent interactions, which are prone to dissociate and subject to concentration-dependent nonspecific aggregation. Heterodimers crosslinked with interchain disulfide bonds are more stable, but it represents a formidable challenge for both the computational design of heterodimers and the manipulation of disulfide pairing for heterodimer synthesis and applications. Here, we report the design, synthesis and application of interchain disulfide-bridged peptide heterodimers with mutual orthogonality by combining computational de novo designs with a directed disulfide pairing strategy. These heterodimers can be used as not only scaffolds for generating functional molecules but chemical tools or building blocks for protein labeling and construction of crosslinking hybrids. This study thus opens the door for using this unexplored dimeric structure space for many biological applications.

Molecular Mechanism of Dual Intercalation in Sac7d-DNA Complexation.

Sac7d belongs to the hyperthermophilc chromosomal protein family, and it is very stable with regard to heat and acidic environments. Unlike many other DNA-protein complexes, the present one is a nonspecific complexation where two amino acids (AA), VAL26 and MET29, are found to intercalate into the same base pair of DNA. Here, we have carried out multiple short molecular dynamic simulations to calculate the distribution of nonspecific protein-DNA aggregates to find the most probable state, which was subsequently used to construct the free energy landscape of protein intercalation into DNA. Analysis of trajectories along the minimum free energy path revealed mechanistic details such as rotation of the protein, simultaneous intercalation of two amino acids, and bending/kinking of the DNA. Moreover, the results indicate a strong interdependency between the intercalating amino acids such that the deintercalation of one AA leads to a spontaneous deintercalation of the other.

Tips on Making Tiny Tips: Secrets to Submicron Nanoelectrospray Emitters.

Nanoelectrospray ionization emitters with submicron tip diameters have significant advantages for use in native mass spectrometry, including the ability to produce resolved charge-state distributions for proteins and macromolecular complexes from standard biochemical buffers that contain high concentrations of nonvolatile salts and to prevent nonspecific aggregation that can occur during droplet evaporation. We report on various factors affecting the tip morphology and provide suggestions for producing and using emitters with submicron tips. Effects of pulling parameters for a Sutter Instrument P-87 tip puller on the resulting tip diameter and morphology are shown. The "Pull" parameter has the largest effect on tip diameter, followed by "Velocity", "Pressure", and "Heat", whereas the "Time" parameter has minimal effect beyond a lower threshold. High "Pull" values generate emitters with multiple tapers, whereas high "Velocity" values generate a tip with only a single tapered region. A protocol for producing reproducible emitters in the submicron size range, as well as guidelines and tips for using these emitters with standard biochemical buffers that contain high concentrations of nonvolatile salts, is presented with the aim of expanding their use within the native mass spectrometry community.

Formation of Visible Aggregates between Rolling Circle Amplification Products and Magnetic Nanoparticles as a Strategy for Point-of-Care Diagnostics.

Visual detection of rolling circle amplification products (RCPs) has been achieved by specific aggregation with magnetic nanoparticles. The method presented here reliably generates aggregates in 1.5 h; these are visible to the naked eye in samples containing at least 0.4 fmol of RCPs. In addition, alternate current susceptometry and absorbance spectroscopy have also been used to quantify the amplified products. The specificity of the detection method was tested, and no non-specific aggregation was detected in samples containing up to 20 fmol of non-complementary amplified DNA. This method is a versatile tool for detecting pathogenic DNA in point-of-care diagnostics, with no readout equipment required. However, chips and automated assays can be used in conjugation with the developed method since detection and quantification can be achieved by commercially available readout instruments.

Efficient detection and robust tracking of spermatozoa in microscopic video

Sperm concentration and motility are generally analysed only in the discrete state in microscopic videos. As for sperm nonspecific aggregation areas, it brings difficulties to accurate sperm detection. In this paper, an algorithm for nonspecific aggregates automatic segmentation, detection and tracking of sperm is proposed. A grid model commensurate with the size of a sperm head is created to segment nonspecific aggregation areas. Multi-scale edge function and new energy functional are designed based on the level set method to realize sperm head segmentation. In the sperm tracking stage, we improve the weight condition and the standard of trust flow quantization based on graph theory method, and simplify the sperm tracking to the vertex matching between two frames to solve the matching failure problem of adjacent frames with small space distance. The proposed method achieves accurate segmentation of sperm non-specific aggregation regions, which outperforms the level set methods of LBF and SBGFR. At the same time, our method can real-time calculation of sperm concentration and motility during sperm tracking. It is compared with four state-of-the-art algorithms, and it gives lower tracking error rates, which has potential applications in male fertility field.

Validation of Pre-Transplant Biomarker Measurement and Risk Score for Non-Relapse Mortality after Allogeneic Hematopoietic Cell Transplant (HCT)

Validation of Pre-Transplant Biomarker Measurement and Risk Score for Non-Relapse Mortality after Allogeneic Hematopoietic Cell Transplant (HCT)

Construction and Piezoelectric Properties of a Single-Peptide Nanotube Composed of Cyclic β-peptides with Helical Peptides on the Side Chains.

To develop nanopiezoelectronics, it is necessary to investigate the relationship between the sizes and piezoelectric properties of the material. Peptide nanotubes (PNTs) composed of cyclic β-peptides have been studied as leading candidates for nanopiezoelectric materials. The current drawback of PNTs is aggregation to form a PNT bundle structure due to strong dipole-dipole interactions between PNTs. Here, we report the construction and piezoelectric properties of single PNTs without nonspecific aggregation by side-chain modification of helical peptides. A cyclic tri-β-peptide with a helical peptide was prepared by multiple-step liquid-phase peptide synthesis and assembled into PNTs by the vapor diffusion method. These nanotubes were characterized by polarized light microscopy and Fourier transform infrared (FTIR) spectroscopy. Additionally, atomic force microscopy (AFM) topographic images showed nanotubes with a height of 4 nm, which corresponds to the diameter of a PNT on a gold-coated mica substrate, indicating that a single PNT was prepared successfully. The converted piezoelectric response of a single PNT was determined to be 1.39 ± 0.12 pm/V. This value was consistent with that of a PNT bundle, which reveals that the piezoelectricity of PNTs is induced by deformation of their cyclic skeletons and is independent of the bundled structure. This finding not only demonstrates a new molecular design strategy to construct these smallest piezoelectric biomaterials by controlling the supramolecular hierarchical structures but also provides insights into the correlation between molecular assembly morphology and size-dependent piezoelectric properties.

Molecular detection using aptamer-modified gold nanoparticles with an immobilized DNA brush for the prevention of non-specific aggregation.

Gold nanoparticles (AuNPs) are often used for biosensing. In particular, aptamer-modified AuNPs are often used for colorimetric molecular detection, where target molecule-induced AuNP aggregates can be recognized by a color change from red to blue. However, non-specific aggregation could be induced by various compounds, leading to false-positive results. In this work we employed high-density ssDNA modification on the AuNP surface to prevent non-specific aggregation. The covalently immobilized DNA brush was used as an anchor for an aptamer specific for the target molecule. Herein, as a proof-of-concept study, we demonstrated detection of estradiol (E2), one of the endocrine-disrupting estrogen molecules as a model target, in the presence of antibiotic kanamycin (KN) as a model of co-contaminating compounds that induce non-specific aggregation of AuNPs. We also developed a smartphone dark field microscope (DFM) to visualize AuNP aggregation. Our previous study demonstrated that the observation of light scattering by AuNP aggregates with DFM can be applied for versatile molecular detection. In this work, we could successfully detect E2 with the smartphone DFM, and the results were verified by the results from a conventional benchtop DFM. This study would contribute to the future field applicability of AuNP-based sensors.