Hierarchical superstructure aerogels for in situ biofluid metabolomics.

High-throughput biofluid metabolomics analysis for screening life-threatening diseases is urgently needed. However, the high salt content of biofluid samples, which introduces severe interference, can greatly limit the analysis throughput. Here, a new 3-D interconnected hierarchical superstructure, namely a "plasmonic gold-on-silica (Au/SiO2) double-layered aerogel", integrating distinctive features of an upper plasmonic gold aerogel with a lower inert silica aerogel was successfully developed to achieve in situ separation and storage of inorganic salts in the silica aerogel, parallel enrichment of metabolites on the surface of the functionalized gold aerogel, and direct desorption/ionization of enriched metabolites by the photo-excited gold aerogel for rapid, sensitive, and comprehensive metabolomics analysis of human serum/urine samples. By integrating all these unique advantages into the hierarchical aerogel, multifunctional properties were introduced in the SALDI substrate to enable its effective utilization in clinical metabolomics for the discovery of reliable metabolic biomarkers to achieve unambiguous differentiation of early and advanced-stage lung cancer patients from healthy individuals. This study provides insight into the design and application of superstructured nanomaterials for in situ separation, storage, and photoexcitation of multi-components in complex biofluid samples for sensitive analysis.

µ-PESI-based MS profiling combined with untargeted metabolomics analysis for rapid identification of red wine geographical origin.

BACKGROUND: The commercial value of red wine is strongly linked to its geographical origin. Given the large global market, there is great demand for high-throughput screening methods to authenticate the geographical source of red wine. However, only limited techniques have been established up to now. RESULTS: Herein, a sensitive and robust method, namely probe electrospray ionization mass spectrometry (µ-PESI-MS), was established to achieve rapid analysis at approximately 1.2 min per sample without any pretreatment. A scotch near the needle tip provides a fixed micro-volume for each analysis to achieve satisfactory ion signal reproducibility (RSD < 26.7%). In combination with a machine learning algorithm, 16 characteristic ions were discovered from thousands of detected ions and were utilized for differentiating red wine origin. Among them, the relative abundances of two characteristic metabolites (trigonelline and proline) correlated with geographical conditions (sun exposure and water stress) were identified, providing the rationale for differentiation of the geographical origin. CONCLUSION: The proposed µ-PESI-MS-based method demonstrates a promising high-throughput determination capability in red wine traceability.

Serum Lipidomic Fingerprints Encode Early Diagnosis and Staging of Lung Cancer on a Novel PbS/Au-Layered Substrate.

Lung cancer (LC) is a major cause of mortality among malignant tumors. Early diagnosis through lipidomic profiling can improve prognostic outcomes. In this study, a uniform PbS/Au-layered substrate that enhances the laser desorption/ionization process, an interfacial process triggered on the substrate surface upon laser excitation, was designed to efficiently characterize the lipidomic profiles of LC patient serum. By controlling the stacking arrangement and particle sizes of PbS QDs and AuNPs, the optimized substrate promotes the generation of excited electrons and creates an enhanced electric field that polarizes analyte molecules, facilitating ion adduction formation ([M + Na]+ and [M + K]+) and enhancing detection sensitivity down to the femtomole level. Combining multivariate statistics and machine learning, a distinct lipidomic biomarker panel is successfully identified for the early diagnosis and staging of LC, with an accurate prediction validated by an area under the curve of 0.9479 and 0.9034, respectively. We also found that 18 biomarkers were significantly correlated with six metabolic pathways associated with LC. These results demonstrate the potential of this innovative PbS/Au-layered substrate as a sensitive platform for accurate diagnosis of LC and facilitate the development of lipidomic-based diagnostic tools for other cancers.

Integration of Non-targeted Proteomics Mass Spectrometry with Machine Learning for Screening Cooked Beef Adulterated Samples.

The degradation of ingredients in heat-processed meat products makes their authentication challenging. In this study, protein profiles of raw beef, chicken, duck, pork, and binary simulated adulterated beef samples (chicken-beef, duck-beef, and pork-beef) and their heat-processed samples were obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Heat-stable characteristic proteins were found by screening the overlapping characteristic protein ion peaks of the raw and corresponding heat-processed samples, which were discovered by partial least-squares discriminant analysis. Based on the 36 heat-stable characteristic proteins, qualitative classification for the raw and heat-processed meats was achieved by extreme gradient boosting. Moreover, quantitative analysis via partial least squares regression was applied to determine the adulteration ratio of the simulated adulterated beef samples. The validity of the approach was confirmed by a blind test with the mean accuracy of 97.4%. The limit of detection and limit of quantification of this method were determined to be 5 and 8%, respectively, showing its practical aspect for the beef authentication.

Covalent Bonding and Coulomb Repulsion-Guided AuNP Array: A Tunable and Reusable Substrate for Metabolomic Characterization of Lung Cancer Patient Sera.

Surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) has gained increased attention in the metabolic characterization of human biofluids. However, the stability and reproducibility of nanoparticle-based substrates remain two of the biggest challenges in high-salt environments. Here, by controlling the extent of Coulomb repulsion of 26 nm positively charged AuNPs, a homogeneous layer of covalently bonded AuNPs on a coverslip with tunable interparticle distances down to 16 nm has been successfully fabricated to analyze small biomolecules in human serum. Compared with the self-assembled AuNP array, the covalently bonded AuNP array showed superior performances on stability, reproducibility, and sensitivity in high-salt environments. The stable attachment of AuNPs maintained a detection reproducibility with a RSD less than 12% and enabled the reusability of the array for 10 experiments without significant signal deterioration (<15%) and carryover effects. Moreover, the closely positioned AuNPs allowed the coupling of photoinduced plasmons to generate an enhanced electric field, which promotes the generation of excited electrons to facilitate the desorption/ionization processes instead of the heat dissipation, thus enhancing the detection sensitivity with detection limits down to the femtomole level. Combined with machine learning methods, the AuNP array has been successfully applied to discover seven biomarkers for differentiating early-stage lung cancer patients from healthy controls. It is anticipated that this simple approach of developing robust AuNP arrays can also be extended to other types of NP arrays for wider applications of SALDI-MS technology.

Sensitive photoelectrochemical detection of colitoxin DNA based on NCDs@CuO/ZnO heterostructured nanocomposites with efficient separation capacity of photo-induced carriers.

A metal-organic framework (MOF) of Cu-TPA (terephthalic acid) microsphere was prepared, followed by calcinating the MOF precursor of Cu-TPA/ZIF-8 mixture to obtain the CuO/ZnO. N-doped carbon dots (NCDs) were employed to combine the CuO/ZnO composite to form a tripartite heterostructured architecture of NCDs@CuO/ZnO, which led to a fierce enlargement of the photocurrent response. This  was ascribed to the thinner-shell structure of the CuO microsphere and the fact that hollow ZnO particles could sharply promote the incidence intensity of visible light. The more porous defectiveness exposed on CuO/ZnO surface was in favor of rapidly infiltrating electrolyte ions. The p-n type CuO/ZnO composite with more contact interface could abridge the transfer distance of photo-induced electron (e-1)/hole (h+) pairs and repress their recombination availably. NCDs not only could boost electron transfer rate on the electrode interface but also successfully sensitized the CuO/ZnO composite, which resulted in high conversion efficiency of photon-to-electron. The probe DNA (S1) was firmly assembled on the modified ITO electrode surface (S1/NCDs@CuO/ZnO) through an amidation reaction. Under optimal conditions, the prepared DNA biosensor displayed a wide linear range of 1.0 × 10-6 ~ 7.5 × 10-1 nM and a low limit of detection (LOD) of 1.81 × 10-7 nM for colitoxin DNA (S2) measure, which exhibited a better photoelectrochemistry (PEC) analysis performance than that obtained by differential pulse voltammetry techniques. The relative standard deviation (RSD) of the sensing platform for target DNA detection of 5.0 × 10-2 nM was 6.3%. This proposed DNA biosensor also showed good selectivity, stability, and reproducibility, demonstrating that the well-designed and synthesized photoactive materials of NCDs@CuO/ZnO are promising candidates for PEC analysis.

Combining MALDI-MS with machine learning for metabolomic characterization of lung cancer patient sera.

An increasing amount of evidence has proven that serum metabolites can instantly reflect disease states. Therefore, sensitive and reproducible detection of serum metabolites in a high-throughput manner is urgently needed for clinical diagnosis. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a high-throughput platform for metabolite detection, but it is hindered by significant signal fluctuations because of the "sweet spot" effect of organic matrices. Here, by screening two transformation methods and four normalization techniques to reduce the significant signal fluctuations of the DHB matrix, an integrated MALDI-MS data processing approach combined with machine learning methods was established to reveal metabolic biomarkers of lung cancer. In our study, 13 distinctive features with statistically significant differences (p < 0.001) between 34 lung cancer patients and 26 healthy controls were selected as significant potential biomarkers of lung cancer. 6 out of the 13 distinctive features were identified as intact metabolites. Our results demonstrate the potential for clinical application of MALDI-MS in serum metabolomics for biomarker screening in lung cancer.

Arsenic trioxide targets Hsp60, triggering degradation of p53 and survivin.

The mechanisms of action of arsenic trioxide (ATO), a clinically used drug for the treatment of acute promyelocytic leukemia (APL), have been actively studied mainly through characterization of individual putative protein targets. There appear to be no studies at a system level. Herein, we integrate metalloproteomics through a newly developed organoarsenic probe, As-AC (C20H17AsN4O3S2) with quantitative proteomics, allowing 37 arsenic binding and 250 arsenic regulated proteins to be identified in NB4, a human APL cell line. Bioinformatics analysis reveals that ATO disrupts multiple physiological processes, in particular, chaperone-related protein folding and cellular response to stress. Furthermore, we discover heat shock protein 60 (Hsp60) as a vital target of ATO. Through biophysical and cell-based assays, we demonstrate that ATO binds to Hsp60, leading to abolishment of Hsp60 refolding capability. Significantly, the binding of ATO to Hsp60 disrupts the formation of Hsp60-p53 and Hsp60-survivin complexes, resulting in degradation of p53 and survivin. This study provides significant insights into the mechanism of action of ATO at a systemic perspective, and serves as guidance for the rational design of metal-based anticancer drugs.

Harvesting More Energetic Photoexcited Electrons from Closely Packed Gold Nanoparticles.

The characterization of photoexcited electrons on the surface of nanomaterial remains challenging. Herein, laser excitation mass spectrometry combined with a chemical thermometer and electron acceptor has been developed to characterize the energetics and population density of photoexcited electrons transferred from gold nanoparticles (AuNPs). In contrast to laser fluence and bias voltage, the hot spots of closely packed AuNPs play a more significant role in enhancing the average energetics of photoexcited electrons, which can be harvested effectively by the electron acceptor. By harvesting more energetic photoexcited electrons for the desorption and ionization process, it is anticipated that the sensitive detection of biomarkers can be achieved, which is beneficial to metabolomic studies and early disease diagnosis.

Surface optimization of gold nanoparticle mass tags for the sensitive detection of protein biomarkers via immuno-capture LI-MS.

HPV-induced cervical cancer is one of the most lethal cancers. Therefore, the development of a reliable and accurate method for the early diagnosis of HPV infections is highly important. Here, gold nanoparticles (AuNPs) were utilized as mass tags in an immuno-capture LI-MS assay for the detection of HPV marker proteins. Through the optimization of the amount of antibodies and surface charges on AuNPs, high antigen detection efficiency with minimal non-specific binding was achieved. With optimized antibody-conjugated AuNPs, low attomole amount of HPV proteins in HeLa cell lysate was quantified.

Sensitive Detection of Separated Charges in Nanohybrids by Laser Excitation Mass Spectrometry with Tetrabutylammonium Cationic Probe.

Charge separation lays the foundation for photocatalysis and photovoltaics, in which the catalytic/voltaic efficiency is primarily related to the amount of separated charges generated. Yet, direct experimental approaches for the quantification of separated charges are very limited, especially for nanostructures in small quantities. Here, by laser excitation mass spectrometry with tetrabutylammonium as a sensitive probe, the separated charges in gold-metal sulfide core-shell nanostructures are determined and correlated with the bandgap of the semiconductor shell. Moreover, the separated charges formed can already be detected unambiguously in only an attomole-level of nanoparticles (i.e., 1 × 108 NPs).

The Hidden Heroes: Holes in Charge-Driven Desorption Mass Spectrometry.

Photoexcited hot carriers have high impacts on various fields. However, in contrast to the attention attracted by the hot-electrons, the contributions of holes are seldom recognized. Yet, by simply removing the hot-electrons but retaining the holes on the surface of photoexcited plasmonic gold nanoparticles, a 60-fold ion intensity amplification could be achieved in laser desorption ionization mass spectrometry. A new desorption mechanism termed "charge-driven desorption" has been derived to rationalize the significant improvement.

Plasmonic gold nanoparticles as multifaceted probe for tissue imaging.

The localized surface plasmon resonance property of gold nanoparticles enables their application as a versatile and sensitive imaging probe, with intense colour, enhanced fluorescence and strong MS ion intensity for biological tissue imaging.

Plasmonic metal nanoparticles as efficient mass tags for ion signal amplification and ultrasensitive detection of protein markers.

The development of sensitive and specific analytical methods is critical for the discovery of molecular biomarkers, which assists disease diagnosis and understanding biological processes. Herein, a highly sensitive method is developed using antibody-conjugated plasmonic metal nanoparticles for the detection of targeted biomarkers down to low attomole level via coupling of immunoassay techniques with laser ionization mass spectrometry (LI-MS). The conjugated antibodies target specific antigens, while the metal nanoparticles act as mass tags and ion reservoirs for the signal amplification. With the characteristic localized surface plasmon resonance (LSPR) properties, gold (AuNPs) and silver nanoparticles (AgNPs) undergo explosive ionization upon laser irradiation to generate abundant characteristic mass reporter ions for strong MS signal amplification. With the antibody-conjugated NPs, detection of trace proteins in various biological samples with complex matrix environment, including urine, cell lysates, and animal tissues was demonstrated.

Silver-gold alloy nanoparticles as tunable substrates for systematic control of ion-desorption efficiency and heat transfer in surface-assisted laser desorption/ionization.

Systematically controlling heat transfer in the surface-assisted laser desorption/ionization (SALDI) process and thus enhancing the analytical performance of SALDI-MS remains a challenging task. In the current study, by tuning the metal contents of Ag-Au alloy nanoparticle substrates (AgNPs, Ag55Au45NPs, Ag15Au85NPs and AuNPs, ∅: ∼2.0 nm), it was found that both SALDI ion-desorption efficiency and heat transfer can be controlled in a wide range of laser fluence (21.3 mJ cm-2 to 125.9 mJ cm-2). It was discovered that ion detection sensitivity can be enhanced at any laser fluence by tuning up the Ag content of the alloy nanoparticle, whereas the extent of ion fragmentation can be reduced by tuning up the Au content. The enhancement effect of Ag content on ion desorption was found to be attributable to the increase in laser absorption efficiency (at 355 nm) with Ag content. Tuning the laser absorption efficiency by changing the metal composition was also effective in controlling the heat transfer from the NPs to the analytes. The laser-induced heating of Ag-rich alloy NPs could be balanced or even overridden by increasing the Au content of NPs, resulting in the reduction of the fragmentation of analytes. In the correlation of experimental measurement with molecular dynamics simulation, the effect of metal composition on the dynamics of the ion desorption process was also elucidated. Upon increasing the Ag content, it was also found that phase transition temperatures, such as melting, vaporization and phase explosion temperature, of NPs could be reduced. This further enhanced the desorption of analyte ions via phase-transition-driven desorption processes. The significant cooling effect on the analyte ions observed at high laser fluence was also determined to be originated from the phase explosion of the NPs. This study revealed that the development of alloy nanoparticles as SALDI substrates can constitute an effective means for the systematic control of ion-desorption efficiency and the extent of heat transfer, which could potentially enhance the analytical performance of SALDI-MS.

Chemical Printing of Biological Tissue by Gold Nanoparticle-Assisted Laser Ablation.

A chemical printing method based on gold nanoparticle (AuNP)-assisted laser ablation has been developed. By rastering a thin layer of AuNPs coated on a rat kidney tissue section with a UV laser, biomolecules are extracted and immediately transferred/printed onto a supporting glass substrate. The integrity of the printed sample is preserved, as revealed by imaging mass spectrometric analysis. By studying the mechanism of the extraction/printing process, transiently molten AuNPs were found to be involved in the process, as supported by the color and morphological changes of the AuNP thin film. The success of this molecular printing method was based on the efficient laser-nanomaterial interaction, that is, the strong photoabsorption, laser-induced heating, and phase-transition properties of the AuNPs. It is anticipated that the molecular printing method can be applied to perform site-specific printing, which extracts and transfers biochemicals from different regions of biological tissue sections to different types of supporting materials for subsequent biochemical analysis with the preservation of the original tissue samples.

Enhancement of Image Contrast, Stability, and SALDI-MS Detection Sensitivity for Latent Fingerprint Analysis by Tuning the Composition of Silver-Gold Nanoalloys.

Metal alloy nanoparticles (NPs) offer a new combination of unique physicochemical properties based on their pure counterparts, which can facilitate the development of novel analytical methods. Here, we demonstrated that Ag-Au alloy NPs could be utilized for optical and mass spectrometric imaging of latent fingerprints (LFPs) with improved image contrast, stability, and detection sensitivity. Upon deposition of Ag-Au alloy NPs (Ag:Au = 60:40 wt %), ridge regions of the LFP became amber colored, while the groove regions appeared purple-blue. The presence of Au in the Ag-Au alloy NPs suppressed aggregation behavior compared to pure AgNPs, thus improving the stability of the developed LFP images. In addition, the Ag component in the Ag-Au alloy NPs enhanced optical absorption efficiency compared to pure AuNPs, resulting in higher contrast LFP images. Moreover, varying the Ag-Au ratio could enable the tuning of the resulting surface plasmonic resonance absorption and hence affect image contrast. Furthermore, the Ag-Au alloy NPs assisted the surface-assisted laser desorption/ionization MS analysis of chemical and biochemical compounds in LFPs, with better detection sensitivity than either pure AgNPs or AuNPs.

Gold nanoparticles bridging infra-red spectroscopy and laser desorption/ionization mass spectrometry for direct analysis of over-the-counter drug and botanical medicines.

With a coating of gold nanoparticles (AuNPs), over-the-counter (OTC) drugs and Chinese herbal medicine granules in KBr pellets could be analyzed by Fourier Transform Infra-red (FT-IR) spectroscopy and Surface-assisted Laser Desorption/Ionization mass spectrometry (SALDI-MS). FT-IR spectroscopy allows fast detection of major active ingredient (e.g., acetaminophen) in OTC drugs in KBr pellets. Upon coating a thin layer of AuNPs on the KBr pellet, minor active ingredients (e.g., noscapine and loratadine) in OTC drugs, which were not revealed by FT-IR, could be detected unambiguously using AuNPs-assisted LDI-MS. Moreover, phytochemical markers of Coptidis Rhizoma (i.e. berberine, palmatine and coptisine) could be quantified in the concentrated Chinese medicine (CCM) granules by the SALDI-MS using standard addition method. The quantitative results matched with those determined by high-performance liquid chromatography with ultraviolet detection. Being strongly absorbing in UV yet transparent to IR, AuNPs successfully bridged FT-IR and SALDI-MS for direct analysis of active ingredients in the same solid sample. FT-IR allowed the fast analysis of major active ingredient in drugs, while SALDI-MS allowed the detection of minor active ingredient in the presence of excipient, and also quantitation of phytochemicals in herbal granules.

Rapid labeling of intracellular His-tagged proteins in living cells.

Small molecule-based fluorescent probes have been used for real-time visualization of live cells and tracking of various cellular events with minimal perturbation on the cells being investigated. Given the wide utility of the (histidine)6-Ni(2+)-nitrilotriacetate (Ni-NTA) system in protein purification, there is significant interest in fluorescent Ni(2+)-NTA-based probes. Unfortunately, previous Ni-NTA-based probes suffer from poor membrane permeability and cannot label intracellular proteins. Here, we report the design and synthesis of, to our knowledge, the first membrane-permeable fluorescent probe Ni-NTA-AC via conjugation of NTA with fluorophore and arylazide followed by coordination with Ni(2+) ions. The probe, driven by Ni(2+)-NTA, binds specifically to His-tags genetically fused to proteins and subsequently forms a covalent bond upon photoactivation of the arylazide, leading to a 13-fold fluorescence enhancement. The arylazide is indispensable not only for fluorescence enhancement, but also for strengthening the binding between the probe and proteins. Significantly, the Ni-NTA-AC probe can rapidly enter different types of cells, even plant tissues, to target His-tagged proteins. Using this probe, we visualized the subcellular localization of a DNA repair protein, Xeroderma pigmentosum group A (XPA122), which is known to be mainly enriched in the nucleus. We also demonstrated that the probe can image a genetically engineered His-tagged protein in plant tissues. This study thus offers a new opportunity for in situ visualization of large libraries of His-tagged proteins in various prokaryotic and eukaryotic cells.

Direct analysis of large living organism by megavolt electrostatic ionization mass spectrometry.

A new ambient ionization method allowing the direct chemical analysis of living human body by mass spectrometry (MS) was developed. This MS method, namely Megavolt Electrostatic Ionization Mass Spectrometry, is based on electrostatic charging of a living individual to megavolt (MV) potential, illicit drugs, and explosives on skin/glove, flammable solvent on cloth/tissue paper, and volatile food substances in breath were readily ionized and detected by a mass spectrometer.