Orders Of Magnitude Dynamic Range Research Articles

Target Recognition Triggered Split DNAzyme based Colorimetric Assay for Direct and Sensitive Methicillin-Resistance Analysis of Staphylococcus aureus.

The accurate and rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) holds significant clinical importance. This work presents a new method for detecting methicillin-resistant Staphylococcus aureus (S. aureus) in clinical samples. The method uses an aptamer-based colorimetric assay that combines a recognizing probe to identify the target and split DNAzyme to amplify the signal, resulting in a highly sensitive and direct analysis of methicillin-resistance. The identification of the PBP2a protein on the membrane of S. aureus in clinical samples leads to the allosterism of the recognizing probe, and thus provides a template for the proximity ligation of split DNAzyme. The proximity ligation of split DNAzyme forms an intact DNAzyme to identify the loop section in the L probe and generates a nicking site to release the loop sequence ("3" and "4" fragments). The "3" and "4" fragments forms an intact sequence to induce the catalytic hairpin assembly, exposing the G-rich section. The released the G-rich sequence of LR probe induces the formation of G-quadruplex-hemin DNAzyme as a colorimetric signal readout. The absorption intensity demonstrated a strong linear association with the logarithm of the S. aureus concentration across a wide range of 5 orders of magnitude dynamic range under the optimized experimental parameters. The limit of detection was calculated to be 23 CFU/ml and the method showed high selectivity for MRSA.

Cover Feature: Enhanced Chemiluminescence of a Superior Luminol Derivative Provides Sensitive Smartphone‐Based Point‐of‐Care Testing with Enzymatic μPAD (Anal. Sens. 4/2023)

The cover image illustrates the principle of a newly developed chemiluminescence (CL) microfluidic paper-based analytical device (μPAD) that enables highly sensitive detection of any catalytic process in which H2O2 is produced. Through the use of a new, sensitive CL luminophore cell phone camera detection allows quantification on-site with the same sensitivity as afforded through a CCD camera in the lab. One-step reactions to time the catalytic reactions can easily be realized through a moveable barrier if desired. Lactate is detected with μM detection limits and three orders of magnitude dynamic range in sweat, the luminophore reaches a pM detection limit. Cover design by Simone Rink. More information can be found in the Research Article by Antje Baeumner and co-workers .

Portable Digital Linear Ion Trap Mass Spectrometer Based on Separate-Region Corona Discharge Ionization Source for On-Site Rapid Detection of Illegal Drugs.

As narcotic control has become worse in the past decade and the death toll of drug abuse hits a record high, there is an increasing demand for on-site rapid detection of illegal drugs. This work developed a portable digital linear ion trap mass spectrometer based on separate-region corona discharge ionization source to meet this need. A separate design of discharge and reaction regions was adopted with filter air as both carrier gas for the analyte and protection of the corona discharge needle. The linear ion trap was driven by a digital waveform with a low voltage (±100 V) to cover a mass range of 50–500 Da with a unit resolution at a scan rate of 10,000 Da/s. Eighteen representative drugs were analyzed, demonstrating excellent qualitative analysis capability. Tandem mass spectrometry (MS/MS) was also performed by ion isolation and collision-induced dissociation (CID) with air as a buffer gas. With cocaine as an example, over two orders of magnitude dynamic range and 10 pg of detection limit were achieved. A single analysis time of less than 10 s was obtained by comparing the information of characteristic ions and product ions with the built-in database. Analysis of a real-world sample further validated the feasibility of the instrument, with the results benchmarked by GC-MS. The developed system has powerful analytical capability without using consumables including solvent and inert gas, meeting the requirements of on-site rapid detection applications.

Enzyme-free dual-amplification assay for colorimetric detection of tetracycline based on Mg2+-dependent DNAzyme assisted catalytic hairpin assembly.

Detection of ultralow concentration of tetracycline (TC) plays a key role in food safety and human health. Herein, we fabricated an enzyme-free dual-amplification assay for sensitive detection of TC in milk. The sensing system ingeniously combined Mg2+-dependent DNAzyme (MNAzyme) cleavage and catalytic hairpin assembly (CHA). Through the binding of TC and specific aptamer (Apt), DNA1 was released from the Apt-DNA1 complex. Then the separated DNA1 would hybridize with DNA2 to activate the catalytic activity of MNAzyme, which subsequently cleaved the substrate H0 and generated a new unit to trigger the following CHA reaction between H1 and H2, thereby releasing the G-rich sequence of H1 to induce the formation of G-quadruplex-hemin DNAzyme as a colorimetric signal readout. Under optimal conditions, the absorption intensity exhibited a significant linear correlation with the logarithm of the target TC concentration over a range of 4 orders of magnitude dynamic range from 1 pM to 10nM. The limit of detection was calculated to be 0.89 pM and the method showed high selectivity for TC. More impressively, the proposed method was employed for TC determination in the milk sample.

MEMS GC Column Performance for Analyzing Organics and Biological Molecules for Future Landed Planetary Missions

We present a novel, innovative approach to gas chromatography-mass spectrometry (GC-MS) based on micro-electro-mechanical systems (MEMS) columns that improve the current, state-of-the-art by dramatically reducing the size, mass, and power resources for deploying GC for future landed missions. The outlet of the MEMS GC column was coupled to a prototype of the MAss Spectrometer for Planetary EXploration (MASPEX) through a heated transfer line into the ion source. MEMS GC-MS experiments were performed to demonstrate linearity of response and establish limit of detection (LOD) to alkanes (organics), fatty acid methyl esters (FAMEs) and chemically derivatized amino acids (biological molecules). Linearity of response to each chemical family was demonstrated over two orders of magnitude dynamic range and limit of detection (LOD) values were single to tens (4–43) of picomoles per 1 μl injection volume. MEMS GC column analytical performance was also demonstrated for a “Mega Mix” of chemical analytes including organics and biological molecules. Chromatographic resolution exceeded 200, retention time reproducibility was << 1% RSD (majority ≤ 0.3%), and peak capacity values calculated to be 124 ± 2 over a 435 s retention time window. The 5.5 m MEMS column was also shown to be a suitable alternative to traditional commercial columns for use in comprehensive two-dimensional gas chromatography (GC × GC). Mass spectra collected from MASPEX showed close consistency with National Institute of Technology (NIST) reference mass spectra and were used for high confidence identification of all eluting analytes.

3-D printed injection system for capillary electrophoresis.

Commercial systems for capillary electrophoresis are designed for the unattended analysis of several samples, and are usually large, complex, and expensive. We report a compact system for manual injection of a single sample in capillary electrophoresis, which is ideal for method development and for student training. The injector consists of two parts that are manufactured by three-dimensional printing (STL and STEP files are included as ESI). One part is immobile and holds an electrode for powering electrophoresis and a gas line for pressurized injection and pumping fluids through the capillary. The second part is removable and is used to hold washing solutions, separation electrolyte, or sample. Conventional machining is used to tap holes to hold the electrode, separation capillary, gas line, and safety interlock. The system is used for either pressure or electrokinetic sample injection, and can be used to pump fluids through the capillary for changing background electrolytes and reconditioning the capillary between runs. We coupled the injection system to our high-dynamic range laser-induced fluorescence detector and evaluated the system by performing capillary zone electrophoresis on solutions of fluorescein. Electrokinetic injection produced a linear response across five orders of magnitude dynamic range (slope of the log-log calibration curve was 1.02), concentration detection limits of 5 pM, and mass detection limits of 1 zmol. Pressure injection produced a linear response across at least four orders of magnitude (slope of the log-log calibration curve was 0.92), concentration detection limits of 2 pM, and mass detection limits of 10 zmol.

Aptamer-Pendant DNA Tetrahedron Nanostructure Probe for Ultrasensitive Detection of Tetracycline by Coupling Target-Triggered Rolling Circle Amplification.

Tetracycline (TET) is a broad-spectrum antibiotic, which is frequently used in the prevention and treatment of animal diseases, feed additives, and so on. However, its residue and accumulation in animal-derived foods could cause several side effects to the human body. Herein, we fabricated TET aptamer-pendant DNA tetrahedral nanostructure-functionalized magnetic beads (Apt-tet MBs) as a probe to detect TET. In the presence of target TET, DNA primer was released from Apt-tet MBs since the TET aptamer could specifically bind TET. Next, the separated DNA primer could effectively initiate rolling circle amplification (RCA) reaction and generate a long tandem single-stranded sequence. Finally, with SYBR Green I as the fluorescence dye, the fluorescence signal could be detected by detection probes through hybridizing the RCA product. Under optimal conditions, the fluorescent signal increased with the increasing target TET concentration within the 5 orders of magnitude dynamic range from 0.001 to 10 ng mL-1. The detection limit was calculated to be 0.724 pg mL-1 and the method showed high selectivity toward TET among different antibiotics. More impressively, this method was employed for TET determination in fish and honey samples. The as-obtained results were consistent with those of ELISA kits, holding great potential in the field of food analysis.

A Simple Approach for Experimental Characterization and Validation of Proton Pencil Beam Profiles

A precise characterization of therapeutic proton pencil beams is essential for commissioning of any treatment planning system (TPS). The dose profile characterization includes measurement of the beam lateral dose profile in the beam core and far from the beam core, in the so called low-dose envelope, and requires a sophisticated detection system with a few orders of magnitude dynamic range. We propose to use a single-quantum sensitive Minipix Timepix detector, along with an in-house designed holder to perform measurements of the pencil beam dose profile in air and in water. We validated the manufacturer calibration of the Minipix Timepix detector in proton beams of various energies and compared the deposited energy spectra to Monte Carlo (MC) simulations. The precision of the lateral dose profile measurements has been systematically validated against Krakow proton facility commissioning data and dose profile simulations performed with MC codes Gate/Geant4 and Fred. We obtained an excellent agreement between Minipix Timepix measurements and simulations demonstrating the feasibility of the system for a simple characterization and validation of proton pencil beams. The proposed approach can be implemented at any proton therapy facility to acquire experimental data needed to commission and validate analytical and MC based TPS.

A Library of Tunable, Portable, and Inducer-Free Promoters Derived from Cyanobacteria.

Cyanobacteria are emerging as hosts for various biotechnological applications. The ability to engineer these photosynthetic prokaryotes greatly depends on the availability of well-characterized promoters. Inducer-free promoters of a range of activities may be desirable for the eventual large-scale, outdoor cultivations. Further, several native promoters of cyanobacteria are repressed by high carbon dioxide or light, and it would be of interest to alter this property. We started with PrbcL and PcpcB, the well-characterized native promoters of the model cyanobacterium Synechococcus elongatus PCC 7942, found upstream of the two abundantly expressed genes, Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase, and phycocyanin β-1 subunit, respectively. The library of 48 promoters created via error-prone PCR of these 300-bp-long native promoters showed 2 orders of magnitude dynamic range with activities that were both lower and higher than those of the wild-type promoters. A few mutants of the PrbcL showed greater strength than PcpcB, which is widely considered a superstrong promoter. A number of mutant promoters did not show repression by high CO2 or light, typically found for PrbcL and PcpcB, respectively. Further, the wild-type and mutant promoters showed comparable activities in the fast-growing and stress-tolerant strains S. elongatus PCC 11801 and PCC 11802, suggesting that the library can be used in different cyanobacteria. Interestingly, the majority of the promoters showed strong expression in E. coli, thus adding to the repertoire of inducer-free promoters for this heterotrophic workhorse. Our results have implications in the metabolic engineering of cyanobacteria and E. coli.

Absolute quantitative analysis of intact and oxidized amino acids by LC-MS without prior derivatization

The precise characterization and quantification of oxidative protein damage is a significant challenge due to the low abundance, large variety, and heterogeneity of modifications. Mass spectrometry (MS)-based techniques at the peptide level (proteomics) provide a detailed but limited picture due to incomplete sequence coverage and imperfect enzymatic digestion. This is particularly problematic with oxidatively modified and cross-linked/aggregated proteins. There is a pressing need for methods that can quantify large numbers of modified amino acids, which are often present in low abundance compared to the high background of non-damaged amino acids, in a rapid and reliable fashion. We have developed a protocol using zwitterionic ion-exchange chromatography coupled with LC-MS to simultaneously quantify both parent amino acids and their respective oxidation products. Proteins are hydrolyzed with methanesulfonic acid in the presence of tryptamine and purified by strong cation exchange solid phase extraction. The method was validated for the common amino acids (excluding Gln, Asn, Cys) and the oxidation products 3-chlorotyrosine (3-ClTyr), 3-nitrotyrosine (3-NO2Tyr), di-tyrosine, Nε-(1-carboxymethyl)-l-lysine, o,o’-di-tyrosine, 3,4,-dihydroxyphenylalanine, hydroxy-tryptophan and kynurenine. Linear standard curves were observed over ~3 orders of magnitude dynamic range (2–1000 pmol for parent amino acids, 80 fmol–20 pmol for oxidation products) with limit-of-quantification values as low as 200 fmol (o,o’-di-tyrosine). The validated method was used to quantify Tyr and Trp loss, and formation of 3-NO2Tyr on the isolated protein anastellin treated with peroxynitrous acid, and for 3-ClTyr formation (over a 2 orders of magnitude range) in cell lysates and complex protein mixtures treated with hypochlorous acid.

HBN-Encapsulated, Graphene-based, Room-temperature Terahertz Receivers, with High Speed and Low Noise

Uncooled terahertz photodetectors (PDs) showing fast (ps) response and high sensitivity (noise equivalent power (NEP) < nW/Hz1/2) over a broad (0.5-10 THz) frequency range are needed for applications in high-resolution spectroscopy (relative accuracy ∼10-11), metrology, quantum information, security, imaging, optical communications. However, present terahertz receivers cannot provide the required balance between sensitivity, speed, operation temperature, and frequency range. Here, we demonstrate uncooled terahertz PDs combining the low (∼2000 kB μm-2) electronic specific heat of high mobility (>50 000 cm2 V-1 s-1) hexagonal boron nitride-encapsulated graphene, with asymmetric field enhancement produced by a bow-tie antenna, resonating at 3 THz. This produces a strong photo-thermoelectric conversion, which simultaneously leads to a combination of high sensitivity (NEP ≤ 160 pW Hz-1/2), fast response time (≤3.3 ns), and a 4 orders of magnitude dynamic range, making our devices the fastest, broad-band, low-noise, room-temperature terahertz PD, to date.

CRISPR/Cas12a-Mediated Interfacial Cleaving of Hairpin DNA Reporter for Electrochemical Nucleic Acid Sensing.

A rapid and sensitive isothermal method is crucial for point-of-care (POC) nucleic acid testing. Recently, RNA-guided CRISPR/Cas12a proteins were discovered to exhibit target-triggered nonspecific single-stranded deoxyribonuclease (ssDNase) activity. Herein, the ssDNase cleavage capacity of the CRISPR/Cas12a system for interfacial hairpin DNA (hpDNA) and linear DNA was investigated in detailed. A novel electrochemical DNA biosensor was then developed via target-induced Cas12a cleaving interfacial hpDNA. In this strategy, the RNA-guided target DNA binding activates the robust Cas12a ssDNase activity. The immobilized hpDNA electrochemical reporters with a low surface coverage and incompact morphological structure present accessible substrates for highly efficient Cas12a cleavage, leading to a highly sensitive electrochemical DNA biosensor. Under the optimal conditions, as low as 30 pM target DNA was detected in about 60 min with 3.5 orders of magnitude dynamic range from 50 pM to 100 nM. Furthermore, the practical application ability of the established sensing method for detecting the target in complex matrices was also demonstrated. The proposed strategy enables rapid and sensitive DNA determination, providing a potential tool for POC molecular diagnostics.

Deuterium analysis by inductively coupled plasma mass spectrometry using polyatomic species: An experimental study supported by plasma chemistry modeling

A new analytical method is proposed for the determination of deuterium (D) by ICP-MS. The method is based on the use of the signal from hydrogen-containing polyatomic ions formed in the inductively coupled plasma. Prior to analytical experiments, a theoretical study was performed to assess the concentration of polyatomic species present in an equilibrium Ar-O-D-H plasma, as a function of temperature and stoichiometric composition. It was established that the highest sensitivity and linearity measurement of D concentration in a wide range can be achieved by monitoring the ions of D2 and ArD, at masses 4 and 42, respectively. Results of the calculations are in good agreement with the experiments. Signal stability, spectral interferences, as well as the effect of plasma parameters were also assessed. Under optimized conditions, the limit of detection (LOD) was found to be 3 ppm atom fraction for deuterium when measured as ArD (in calcium and potassium free water), or 78 ppm when measured as D2. The achieved LOD values and the 4 to 5 orders of magnitude dynamic range easily allow the measurement of deuterium concentrations at around or above the natural level, up to nearly 100% (or 1 Mio ppm) in a standard quadrupole ICP-MS instrument. An even better performance is expected from the method in high resolution ICP-MS instruments equipped with low dead volume sample introduction systems.

Positioning of space objects by laser-induced jets

Laser-induced thrust provides a number of significant advantages over the currently used methods: virtually any material can be used as a working medium; radiation source and its power unit can be located outside the spacecraft; it is possible to provide a minimum impulse bit of 1 nN s or less; momentum imparted at single impact can be controlled within 2 orders of magnitude dynamic range. We have considered recoil momentum generation at femtosecond to continuous laser impact range on different materials normalized by laser output performance to evaluate momentum coupling to on-board energy system. It is shown that better momentum coupling at short wavelength is not worth of associated energy losses, but laser pulse shortening to picosecond range is. Data reported here on laser thrust generation efficiency and methods of laser impact layout are of interest not for small spacecraft application range broadening only, but also for the prevention of emergency situations development (launch to unplanned orbit, uncontrolled rotation, etc.), space debris removal, and anti-asteroid protection of the Earth – possible impact layouts for such missions are considered.

Transistor-based immunosensing in human serum samples without on-site calibration

Health monitoring and disease diagnostics are increasingly moving away from central laboratories towards the point of need. Despite tremendous progress in transducer research, the general need for sample preparation and calibration has impeded wide-spread adoption of new biosensor technologies. In this work, we realize an on-site calibration-free biosensor by using “differential” readout of pairs of differently functionalized field-effect transistors (FET). Specifically, biosensor 1 carries capture antibodies for the target of interest while biosensor 2 is passivated with a blocking agent. We found that their real-time signal difference is largely unaffected by non-specific binding, drift or device-to-device variations. Using this system, highly sensitive detection of a clinically relevant model antigen, thyroid-stimulating hormone, is demonstrated. The sensors are validated in human serum samples vs. a state-of-the-art central laboratory analyzer. The FET-based platform reaches over 4 orders of magnitude dynamic range with a sub-picomolar lower detection limit and an upper detection limit superior to the state-of-the-art system (>10 nM vs. ˜0.1 nM). In contrast to existing technology, the FET sensors do not require any expensive optical components, labelled detection antibodies, sample pre-treatment or washing steps, and remove the need for sensor calibration. These results represent a critical step towards clinical testing outside the centralized lab.

Gas chromatography with tandem cold electron ionization mass spectrometric detection and vacuum ultraviolet detection for the comprehensive analysis of fentanyl analogues

Fentanyl and its derivatives are amongst the ever-growing list of emerging drugs which are impinging on current traditional analytical techniques employed in forensic laboratories. To avoid current regulations, fentanyl analogues are being illicitly synthesized by slight alterations of functional groups to the fentanyl skeleton leading to inaccurate identifications, thus posing the greatest challenge to laboratories. In this study, a novel analytical technique is presented in which gas chromatography (GC) is interfaced with both cold electron ionization mass spectrometric (cold EI MS) and vacuum ultraviolet (VUV) detection by the means of a flow splitter for the simultaneous qualitative and quantitative analysis of twenty-four fentanyl analogues, including seven sets of positional isomers. For most of the twenty-four analogues, enhanced molecular ions were obtained with at least 1% intensity relative to base peak. In addition to enhanced molecular ions, the GC-cold EI MS maintained fragmentation pathways observed by GCMS with classical electron ionization. For the most part, VUV detection resulted in unique VUV spectra for fentanyl analogues including positional isomers. The combination of these two complementary detectors in tandem with the high resolving power of the gas chromatograph, allows for higher confidence in analyte identification by the combination of retention times, cold EI mass spectra and VUV spectra. The preferred method for quantitation was based on VUV detection and offered excellent determination coefficients (R2≥0.999) for most analytes over two orders of magnitude dynamic range, without the need for deuterated internal standards. For both run-to-run and day-to-day repeatability studies, at moderate solute concentrations, the correct fentanyl related compound was identified in almost every instance from a library containing all the fentanyl analogues plus hundreds of other analytes.

Quantitation of 87 Proteins by nLC-MRM/MS in Human Plasma: Workflow for Large-Scale Analysis of Biobank Samples.

A multiple reaction monitoring (MRM) assay was developed for precise quantitation of 87 plasma proteins including the three isoforms of apolipoprotein E (APOE) associated with cardiovascular diseases using nanoscale liquid chromatography separation and stable isotope dilution strategy. The analytical performance of the assay was evaluated and we found an average technical variation of 4.7% in 4-5 orders of magnitude dynamic range (≈0.2 mg/L to 4.5 g/L) from whole plasma digest. Here, we report a complete workflow, including sample processing adapted to 96-well plate format and normalization strategy for large-scale studies. To further investigate the MS-based quantitation the amount of six selected proteins was measured by routinely used clinical chemistry assays as well and the two methods showed excellent correlation with high significance (p-value < 10e-5) for the six proteins, in addition for the cardiovascular predictor factor, APOB: APOA1 ratio (r = 0.969, p-value < 10e-5). Moreover, we utilized the developed assay for screening of biobank samples from patients with myocardial infarction and performed the comparative analysis of patient groups with STEMI (ST- segment elevation myocardial infarction), NSTEMI (non ST- segment elevation myocardial infarction) and type-2 AMI (type-2 myocardial infarction) patients.

Improved Sensitivity for the Qualitative and Quantitative Analysis of Active Ricin by MALDI-TOF Mass Spectrometry.

Ricin is a highly toxic protein which causes cell death by blocking protein synthesis and is considered a potential bioterrorism agent. Rapid and sensitive detection of ricin toxin in various types of sample matrices is needed as an emergency requirement for public health and antibioterrorism response. An in vitro MALDI TOF MS-based activity assay that detects ricin mediated depurination of synthetic substrates was improved through optimization of the substrate, reaction conditions, and sample preparation. In this method, the ricin is captured by a specific polycolonal antibody followed by hydrolysis reaction. The ricin activity is determined by detecting the unique cleavage product of synthetic oligomer substrates. The detection of a depurinated substrate was enhanced by using a more efficient RNA substrate and optimizing buffer components, pH, and reaction temperature. In addition, the factors involved in mass spectrometry analysis, such as MALDI matrix, plate, and sample preparation, were also investigated to improve the ionization of the depurinated product and assay reproducibility. With optimized parameters, the limit of detection of 0.2 ng/mL of ricin spiked in buffer and milk was accomplished, representing more than 2 orders of magnitude enhancement in assay sensitivity. Improving assay's ruggeddness or reproducibility also made it possible to quantitatively detect active ricin with 3 orders of magnitude dynamic range.

High Photoluminescence Quantum Yield in Band Gap Tunable Bromide Containing Mixed Halide Perovskites.

Hybrid organic-inorganic halide perovskite based semiconductor materials are attractive for use in a wide range of optoelectronic devices because they combine the advantages of suitable optoelectronic attributes and simultaneously low-cost solution processability. Here, we present a two-step low-pressure vapor-assisted solution process to grow high quality homogeneous CH3NH3PbI3-xBrx perovskite films over the full band gap range of 1.6-2.3 eV. Photoluminescence light-in versus light-out characterization techniques are used to provide new insights into the optoelectronic properties of Br-containing hybrid organic-inorganic perovskites as a function of optical carrier injection by employing pump-powers over a 6 orders of magnitude dynamic range. The internal luminescence quantum yield of wide band gap perovskites reaches impressive values up to 30%. This high quantum yield translates into substantial quasi-Fermi level splitting and high "luminescence or optically implied" open-circuit voltage. Most importantly, both attributes, high internal quantum yield and high optically implied open-circuit voltage, are demonstrated over the entire band gap range (1.6 eV ≤ Eg ≤ 2.3 eV). These results establish the versatility of Br-containing perovskite semiconductors for a variety of applications and especially for the use as high-quality top cell in tandem photovoltaic devices in combination with industry dominant Si bottom cells.

Laser desorption ionization (LDI) silicon nanopost array chips fabricated using deep UV projection lithography and deep reactive ion etching

A nanofabricated laser desorption ionization mass spectrometry (LDI-MS) chip for quantitation of small molecules.