Multiplexed Signal Ion Emission Reactive Release Amplification (SIERRA) Assay for the Culture-Free Detection of Gram-Negative and Gram-Positive Bacteria and Antimicrobial Resistance Genes.

The global prevalence of antibiotic-resistant bacteria has increased the risk of dangerous infections, requiring rapid diagnosis and treatment. The standard method for diagnosis of bacterial infections remains dependent on slow culture-based methods, carried out in central laboratories, not easily extensible to rapid identification of organisms, and thus not optimal for timely treatments at the point-of-care (POC). Here, we demonstrate rapid detection of bacteria by combining electrochemical immunoassays (EC-IA) for pathogen identification with confirmatory quantitative mass spectral immunoassays (MS-IA) based on signal ion emission reactive release amplification (SIERRA) nanoparticles with unique mass labels. This diagnostic method uses compatible reagents for all involved assays and standard fluidics for automatic sample preparation at POC. EC-IA, based on alkaline phosphatase-conjugated pathogen-specific antibodies, quantified down to 104 bacteria per sample when testing Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa lysates. EC-IA quantitation was also obtained for wound samples. The MS-IA using nanoparticles against S. aureus, E. coli, Klebsiella pneumoniae, and P. aeruginosa allowed selective quantitation of ∼105 bacteria per sample. This method preserves bacterial cells allowing extraction and amplification of 16S ribosomal RNA genes and antibiotic resistance genes, as was demonstrated through identification and quantitation of two strains of E. coli, resistant and nonresistant due to ß-lactamase cefotaximase genes. Finally, the combined immunoassays were compared against culture using remnant deidentified patient urine samples. The sensitivities for these immunoassays were 83, 95, and 92% for the prediction of S. aureus, P. aeruginosa, and E. coli or K. pneumoniae positive culture, respectively, while specificities were 85, 92, and 97%. The diagnostic platform presented here with fluidics and combined immunoassays allows for pathogen isolation within 5 min and identification in as little as 15 min to 1 h, to help guide the decision for additional testing, optimally only on positive samples, such as multiplexed or resistance gene assays (6 h).

Evidence for Many Unique Solution Structures for Chymotrypsin Inhibitor 2: A Thermodynamic Perspective Derived from vT-ESI-IMS-MS Measurements.

Chymotrypsin inhibitor 2 (CI-2) is a classic model for two-state cooperative protein folding and is one of the most extensively studied systems. Alan Fersht, a pioneer in the field of structural biology, has studied the wild-type (wt) and over 100 mutant forms of CI-2 with traditional analytical and biochemical techniques. Here, we examine wt CI-2 and three mutant forms (A16G, K11A, L32A) to demonstrate the utility of variable-temperature (vT) electrospray ionization (ESI) paired with ion mobility spectrometry (IMS) and mass spectrometry (MS) to map the free energy folding landscape. As the solution temperature is increased, the abundance of each of the six ESI charge states for wt CI-2 and each mutant is found to vary independently. These results require that at least six unique types of CI-2 solution conformers are present. Ion mobility analysis reveals that within each charge state there are additional conformers having distinct solution temperature profiles. A model of the data at ∼30 different temperatures for all four systems suggests the presence of 41 unique CI-2 solution conformations. A thermodynamic analysis of this system yields values of ΔCp as well as ΔG, ΔH, and ΔS for each state at every temperature studied. Detailed energy landscapes derived from these data provide a rare glimpse into Anfinsen's thermodynamic hypothesis and the process of thermal denaturation, normally thought of as a cooperative two-state transition involving the native state and unstructured denatured species. Specifically, as the temperature is varied, the entropies and enthalpies of different conformers undergo dramatic changes in magnitude and relative order to maintain the delicate balance associated with equilibrium.

Utilization of electronic health records for the assessment of adiponectin receptor autoantibodies during the progression of cardio-metabolic comorbidities.

BACKGROUND: Diabetes is a complex, multi-symptomatic disease whose complications drives increases in healthcare costs as the diabetes prevalence grows rapidly world-wide. Real-world electronic health records (EHRs) coupled with patient biospecimens, biological understanding, and technologies can characterize emerging diagnostic autoimmune markers resulting from proteomic discoveries. METHODS: Circulating autoantibodies for C-terminal fragments of adiponectin receptor 1 (IgG-CTF) were measured by immunoassay to establish the reference range using midpoint samples from 1862 participants in a 20-year observational study of type 2 diabetes and cardiovascular arterial disease (CVAD) conducted by the Fairbanks Institute. The White Blood Cell elastase activity in these patients was assessed using immunoassays for Bikunin and Uristatin. Participants were assigned to four cohorts (healthy, T2D, CV, CV+T2D) based on analysis of their EHRs and the diagnostic biomarkers values and patient status were assessed ten-years post-sample. RESULTS: The IgG-CTF reference range was determined to be 75-821 ng/mL and IgG-CTF out-of-range values did not predict cohort or comorbidity as determined from the EHRs at 10 years after sample collection nor did IgG-CTF demonstrate a significant risk for comorbidity or death. Many patients at sample collection time had other conditions (hypertension, hyperlipidemia, or other risk factors) of which only hypertension, Uristatin and Bikunin values correlated with increased risk of developing additional comorbidities (odds ratio 2.58-13.11, P<0.05). CONCLUSIONS: This study confirms that retrospective analysis of biorepositories coupled with EHRs can establish reference ranges for novel autoimmune diagnostic markers and provide insights into prediction of specific health outcomes and correlations to other markers.

Identifying extractable profiles from 3D printed medical devices.

With the ability to create customizable products tailored to individual patients, the use of 3D printed medical devices has rapidly increased in recent years. Despite such interest in these materials, a risk assessment based on the material characterization of final device extracts-as per regulatory guidance-has not yet been completed, even though the printing process may potentially impact the leachability of polymer components. To further our understanding of the chemical impact of 3D printed medical devices, this study investigated the extractable profiles of four different materials, including a PLA polymer advertised as "FDA-approved". The fusion deposition modeling (FDM) printing process created distinct chemical and physical signatures in the extracts of certain materials. The application of an annealing procedure to printed devices led to a substantial decrease in extractable components by as much as a factor of 50. In addition, the use of a brass printing nozzle led to an increase in the amount of Pb detected in 3D printed device extracts. The data generated provides valuable information that can be used to help assess extractable risks of 3D printed medical devices, assist with future 3D printing designs, and may provide insight for agencies tasked with governing 3D printed medical device regulations.

Enumeration of Rare Cells in Whole Blood by Signal Ion Emission Reactive Release Amplification with Same-Sample RNA Analysis.

Herein is presented a platform capable of detecting less than 30 cells from a whole blood sample by size-exclusion filtration, microfluidic sample handling, and mass spectrometric detection through signal ion emission reactive release amplification (SIERRA). This represents an approximate 10-fold improvement in detection limits from previous work. Detection by SIERRA is accomplished through the use of novel nanoparticle reagents coupled with custom fluidic fixtures for precise sample transfer. Sample processing is performed in standardized 96-well microtiter plates with commonly available laboratory instrumentation to facilitate assay automation. The detection system is easily amenable to multiplex detection, and compatibility with PCR-based gene assays is demonstrated.

Ion Separation in Air Using a Three-Dimensional Printed Ion Mobility Spectrometer.

The performance of a small, plastic drift tube ion mobility spectrometer (DT-IMS) is described. The IMS was manufactured using three-dimensional (3D) printing techniques and operates in the open air at ambient pressure, temperature, and humidity. The IMS housing and electrodes were printed from nonconductive polylactic acid (PLA, housing) and conductive polyethylene terephthalate glycol-modified polymer containing multiwalled carbon nanotubes (PETG-CNT, electrodes). Ring electrodes consisting of both an inner disk and an outer ring were used to prevent neutral transmission while maximizing ion transmission. As a stand-alone instrument, the 3D printed IMS is shown to achieve resolving powers of between 24 and 50 in positive ion mode using tetraalkylammonium bromide salts (TAA), benzylamines (mono-, di-, and tri-), and illicit drugs (MA, MDEA, and haloperidol). Resolving powers of between 29 and 42 were achieved in negative ion mode using sodium alkyl sulfates (C8, C12, C16, and C18). Reduced ion mobilities of TAA cations (C2-C8) were calculated at 14% relative humidity in air to be 1.36, 1.18, 1.03, 0.90, 0.80, 0.73, and 0.67, respectively. The effect of humidity on reduced ion mobilities of TAA cations is discussed. 3D printing is shown to be a quick and cost-effective way to produce small IMS instruments that can compete in performance with conventionally manufactured IMS instruments that also operate in the open air. An important difference between this IMS and other instruments is the absence of a counter gas flow.

Tumor Cell Detection by Mass Spectrometry Using Signal Ion Emission Reactive Release Amplification.

A method is presented for the detection of circulating tumor cells (CTC) using mass spectrometry (MS), through reporter-ion amplification. Particles functionalized with short-chain peptides are bound to cells through antibody-antigen interactions. Selective release and MS detection of peptides is shown to detect as few as 690 cells isolated from a 10 mL blood sample. Here we present proof-of-concept results that pave the way for further investigations.

Lipid and metabolite profiles of human brain tumors by desorption electrospray ionization-MS.

Examination of tissue sections using desorption electrospray ionization (DESI)-MS revealed phospholipid-derived signals that differ between gray matter, white matter, gliomas, meningiomas, and pituitary tumors, allowing their ready discrimination by multivariate statistics. A set of lower mass signals, some corresponding to oncometabolites, including 2-hydroxyglutaric acid and N-acetyl-aspartic acid, was also observed in the DESI mass spectra, and these data further assisted in discrimination between brain parenchyma and gliomas. The combined information from the lipid and metabolite MS profiles recorded by DESI-MS and explored using multivariate statistics allowed successful differentiation of gray matter (n = 223), white matter (n = 66), gliomas (n = 158), meningiomas (n = 111), and pituitary tumors (n = 154) from 58 patients. A linear discriminant model used to distinguish brain parenchyma and gliomas yielded an overall sensitivity of 97.4% and a specificity of 98.5%. Furthermore, a discriminant model was created for tumor types (i.e., glioma, meningioma, and pituitary), which were discriminated with an overall sensitivity of 99.4% and a specificity of 99.7%. Unsupervised multivariate statistics were used to explore the chemical differences between anatomical regions of brain parenchyma and secondary infiltration. Infiltration of gliomas into normal tissue can be detected by DESI-MS. One hurdle to implementation of DESI-MS intraoperatively is the need for tissue freezing and sectioning, which we address by analyzing smeared biopsy tissue. Tissue smears are shown to give the same chemical information as tissue sections, eliminating the need for sectioning before MS analysis. These results lay the foundation for implementation of intraoperative DESI-MS evaluation of tissue smears for rapid diagnosis.

Ion creation, ion focusing, ion/molecule reactions, ion separation, and ion detection in the open air in a small plastic device.

A method is presented in which ions are generated and manipulated in the ambient environment using polymeric electrodes produced with a consumer-grade 3D printer. The ability to focus, separate, react, and detect ions in the ambient environment is demonstrated and the data agree well with simulated ion behaviour.

Using ambient ion beams to write nanostructured patterns for surface enhanced Raman spectroscopy.

Electrolytic spray deposition was used to pattern surfaces with 2D metallic nanostructures. Spots that contain silver nanoparticles (AgNP) were created by landing solvated silver ions at desired locations using electrically floated masks to focus the metal ions to an area as little as 20 µm in diameter. The AgNPs formed are unprotected and their aggregates can be used for surface-enhanced Raman spectroscopy (SERS). The morphology and SERS activity of the NP structures were controlled by the surface coverage of landed silver ions. The NP structures created could be used as substrates onto which SERS samples were deposited or prepared directly on top of predeposited samples of interest. The evenly distributed hot spots in the micron-sized aggregates had an average SERS enhancement factor of 10(8) . The surfaces showed SERS activity when using lasers of different wavelengths (532, 633, and 785 nm) and were stable in air.