Metal-Organic Framework Stationary Phases for One- and Two-Dimensional Micro-Gas Chromatographic Separations of Light Alkanes and Polar Toxic Industrial Chemicals.

Despite promising advances with metal-organic frameworks (MOFs) as stationary phases for chromatography, the application of MOFs for one- and two-dimensional micro-gas chromatography (µGC and µGC × µGC) applications has yet to be shown. We demonstrate for the first time, µGC columns coated with two different MOFs, HKUST-1 and ZIF-8, for the rapid separation of high volatility light alkane hydrocarbons (natural gas) and determined the partition coefficients for toxic industrial chemicals, using µGC and µGC × µGC systems. Complete separation of natural gas components, methane through pentane, was completed within 1 min, with sufficient resolution to discriminate n-butane from i-butane. Layer-by-layer controlled deposition cycles of the MOFs were accomplished to establish the optimal film thickness, which was validated using GC (sorption thermodynamics), quartz-crystal microbalance gravimetric analysis and scanning electron microscopy. Complete surface coverage was not observed until after ~17 deposition cycles. Propane retention factors with HKUST-1-coated µGC and a state-of-the-art polar, porous-layer open-tubular (PLOT) stationary phase were approximately the same at ~7.5. However, with polar methanol, retention factors with these two stationary phases were 748 and 59, respectively, yielding methanol-to-propane selectivity factors of ~100 and ~8, respectively, a 13-fold increase in polarity with HKUST-1. These studies advance the applications of MOFs as µGC stationary phase.

A high-speed, high-performance, microfabricated comprehensive two-dimensional gas chromatograph.

A small, consumable-free, low-power, ultra-high-speed comprehensive GC×GC system consisting of microfabricated columns, nanoelectromechanical system (NEMS) cantilever resonators for detection, and a valve-based stop-flow modulator is demonstrated. The separation of a highly polar 29-component mixture covering a boiling point range of 46 to 253 °C on a pair of microfabricated columns using a Staiger valve manifold in less than 7 seconds, and just over 4 seconds after the ensemble holdup time is demonstrated with a downstream FID. The analysis time of the second dimension was 160 ms, and peak widths in the second dimension range from 10-60 ms. A peak capacity of just over 300 was calculated for a separation of just over 6 s. Data from a continuous operation testing over 40 days and 20 000 runs of the GC×GC columns with the NEMS resonators using a 4-component test set is presented. The GC×GC-NEMS resonator system generated second-dimension peak widths as narrow as 8 ms with no discernable peak distortion due to under-sampling from the detector.

Thermodynamic Studies on a Hydrogen Bonded Acidic 3,5-Bis(trifluoromethyl)phenol-functionalized Polymer as a Gas Chromatography Stationary Phase for Selectively Speciating Chemical Warfare Agents.

We describe for the first time hydrogen bonded acid (HBA) polymer, poly{methyl[3-(2-hydroxyl, 4,6-bistrifluoromethyl)phenyl]propylsiloxane}, (DKAP), as stationary phase for gas chromatography (µGC) of organophosphate (OP), chemical warfare agent (CWA) surrogates, dimethylmethylphosphonate (DMMP), diisopropylmethylphosphonate (DIMP), diethylmethylphosphonate (DEMP), and trimethylphosphate (TMP), with high selectivity. Absorption of OPs to DKAP was one-to-several orders of magnitude higher relative to commercial polar, mid-polar, and nonpolar stationary phases. We also present for the first-time thermodynamic studies on the absorption of OP vapors and quantitative binding energy data for interactions with various stationary phases. These data help to identify the best pair of hetero-polar columns for a two-dimensional GC system, employing a nonpolar stationary phase as GC1 and DKAP as the GC2 stationary phase, for selective and rapid field detection of CWAs.

Volatile Metabolites Emission by In Vivo Microalgae-An Overlooked Opportunity?

Fragrances and malodors are ubiquitous in the environment, arising from natural and artificial processes, by the generation of volatile organic compounds (VOCs). Although VOCs constitute only a fraction of the metabolites produced by an organism, the detection of VOCs has a broad range of civilian, industrial, military, medical, and national security applications. The VOC metabolic profile of an organism has been referred to as its 'volatilome' (or 'volatome') and the study of volatilome/volatome is characterized as 'volatilomics', a relatively new category in the 'omics' arena. There is considerable literature on VOCs extracted destructively from microalgae for applications such as food, natural products chemistry, and biofuels. VOC emissions from living (in vivo) microalgae too are being increasingly appreciated as potential real-time indicators of the organism's state of health (SoH) along with their contributions to the environment and ecology. This review summarizes VOC emissions from in vivo microalgae; tools and techniques for the collection, storage, transport, detection, and pattern analysis of VOC emissions; linking certain VOCs to biosynthetic/metabolic pathways; and the role of VOCs in microalgae growth, infochemical activities, predator-prey interactions, and general SoH.

Monitoring and modulating ion traffic in hybrid lipid/polymer vesicles.

Controlling the traffic of molecules and ions across membranes is a critical feature in a number of biologically relevant processes and highly desirable for the development of technologies based on membrane materials. In this paper, ion transport behavior of hybrid lipid/polymer membranes was studied in the absence and presence of ion transfer agents. A pH-sensitive fluorophore was used to investigate ion (H+/OH-) permeability across hybrid lipid/polymer membranes as a function of the fraction of amphiphilic block copolymer. It was observed that vesicles with intermediate lipid/polymer ratios tend to be surprisingly more permeable to ion transport than the pure lipid or pure polymer vesicles. Hybrid vesicle permeability could be further modulated with valinomycin, nigericin, or gramicidin A, which significantly expedite the dissipation of externally-imposed pH gradients by facilitating the transport of the rate-limiting co-ions (e.g. K+) ions across the membrane. For gramicidin A, ion permeability decreased with increasing polymer mole fraction, and the method of introduction of gramicidin A into the membrane played an important role. Strategies to incorporate biofunctional molecules and facilitate their activity in synthetic systems are highly desirable for developing artificial organelles or other synthetic compartmentalized structures requiring control over molecular traffic across biomimetic membranes.

Microfabrication of a gadolinium-derived solid-state sensor for thermal neutrons.

Neutron sensing is critical in civilian and military applications. Conventional neutron sensors are limited by size, weight, cost, portability and helium supply. Here the microfabrication of gadolinium (Gd) conversion material-based heterojunction diodes for detecting thermal neutrons using electrical signals produced by internal conversion electrons (ICEs) is described. Films with negligible stress were produced at the tensile-compressive crossover point, enabling Gd coatings of any desired thickness by controlling the radiofrequency sputtering power and using the zero-point near p(Ar) of 50 mTorr at 100 W. Post-deposition Gd oxidation-induced spallation was eliminated by growing a residual stress-free 50 nm neodymium-doped aluminum cap layer atop Gd. The resultant coatings were stable for at least 6 years, demonstrating excellent stability and product shelf-life. Depositing Gd directly on the diode surface eliminated the air gap, leading to a 200-fold increase in electron capture efficiency and facilitating monolithic microfabrication. The conversion electron spectrum was dominated by ICEs with energies of 72, 132 and 174 keV. Results are reported for neutron reflection and moderation by polyethylene for enhanced sensitivity, and γ- and X-ray elimination for improved specificity. The optimal Gd thickness was 10.4 µm for a 300 µm-thick partially depleted diode of 300 mm2 active surface area. Fast detection (within 10 min) at a neutron source-to-diode distance of 11.7 cm was achieved with this configuration. All ICE energies along with γ-ray and Kα,ß X-rays were modeled to emphasize correlations between experiment and theory. Semi-conductor thermal neutron detectors offer advantages for field-sensing of radioactive neutron sources.

Rapid Nucleic Acid Extraction and Purification Using a Miniature Ultrasonic Technique.

Miniature ultrasonic lysis for biological sample preparation is a promising technique for efficient and rapid extraction of nucleic acids and proteins from a wide variety of biological sources. Acoustic methods achieve rapid, unbiased, and efficacious disruption of cellular membranes while avoiding the use of harsh chemicals and enzymes, which interfere with detection assays. In this work, a miniature acoustic nucleic acid extraction system is presented. Using a miniature bulk acoustic wave (BAW) transducer array based on 36° Y-cut lithium niobate, acoustic waves were coupled into disposable laminate-based microfluidic cartridges. To verify the lysing effectiveness, the amount of liberated ATP and the cell viability were measured and compared to untreated samples. The relationship between input power, energy dose, flow-rate, and lysing efficiency were determined. DNA was purified on-chip using three approaches implemented in the cartridges: a silica-based sol-gel silica-bead filled microchannel, nucleic acid binding magnetic beads, and Nafion-coated electrodes. Using E. coli, the lysing dose defined as ATP released per joule was 2.2× greater, releasing 6.1× more ATP for the miniature BAW array compared to a bench-top acoustic lysis system. An electric field-based nucleic acid purification approach using Nafion films yielded an extraction efficiency of 69.2% in 10 min for 50 µL samples.

Pulsed Discharge Helium Ionization Detector for Highly Sensitive Aquametry.

Trace moisture quantitation is crucial in medical, civilian and military applications. Current aquametry technologies are limited by the sample volume, reactivity, or interferences, and/or instrument size, weight, power, cost, and complexity. We report for the first time on the use of a pulsed discharge helium ionization detector (PDHID-D2) (∼196 cm(3)) for the sensitive (limit of detection, 0.047 ng; 26 ppm), linear (r(2) >0.99), and rapid (< 2 min) quantitation of water using a small (0.2 - 5.0 µL) volume of liquid or gas. The relative humidity sensitivity was 0.22% (61.4 ppmv) with a limit of detection of less than 1 ng moisture with gaseous samples. The sensitivity was 10 to 100 to fold superior to competing technologies without the disadvantages inherent to these technologies. The PDHID-D2, due to its small footprint and low power requirement, has good size, weight, and power-portability (SWAPP) factors. The relatively low cost (∼$5000) and commercial availability of the PDHID-D2 makes our technique applicable to highly sensitive aquametry.

Development of a Mesoscale Pulsed Discharge Helium Ionization Detector for Portable Gas Chromatography.

Miniaturization of gas chromatography (GC) instrumentation enables field detection of volatile organic compounds (VOCs) for chembio-applications such as clandestine human transport and disease diagnostics. We fabricated a mesoscale pulsed discharge helium ionization detector (micro-PDHID) for integrating with our previously described mini-GC hardware. Stainless steel electrodes fabricated by photochemical etching and electroforming facilitated rapid prototyping and enabled nesting of inter-electrode insulators for self-alignment of the detector core during assembly. The prototype was ∼10 cm(3) relative to >400 cm(3) of a commercial PDHID, but with a comparable time to sweep a VOC peak from the detector cell (170 ms and 127 ms, respectively). Electron trajectory modeling, gas flow rate, voltage bias, and GC outlet location were optimized for improving sensitivity. Despite 40-fold miniaturization, the micro-PDHID detected 18 ng of the human emanation, 3-methyl-2-hexenoic acid with <3-fold decrease in sensitivity relative to the commercial detector. The micro-PDHID was rugged and operated for 9 months without failure.

Easy parallel screening of reagent stability, quality control, and metrology in solid phase peptide synthesis (SPPS) and peptide couplings for microarrays.

Evaluating the stability of coupling reagents, quality control (QC), and surface functionalization metrology are all critical to the production of high quality peptide microarrays. We describe a broadly applicable screening technique for evaluating the fidelity of solid phase peptide synthesis (SPPS), the stability of activation/coupling reagents, and a microarray surface metrology tool. This technique was used to assess the stability of the activation reagent 1-{[1-(Cyano-2-ethoxy-2-oxo-ethylidenaminooxy)dimethylamino-morpholinomethylene]}methaneaminiumHexafluorophosphate (COMU) (Sigma-Aldrich, St. Louis, MO, USA) by SPPS of Leu-Enkephalin (YGGFL) or the coupling of commercially synthesized YGGFL peptides to (3-aminopropyl)triethyoxysilane-modified glass surfaces. Coupling efficiency was quantitated by fluorescence signaling based on immunoreactivity of the YGGFL motif. It was concluded that COMU solutions should be prepared fresh and used within 5 h when stored at ~23 °C and not beyond 24 h if stored refrigerated, both in closed containers. Caveats to gauging COMU stability by absorption spectroscopy are discussed. Commercial YGGFL peptides needed independent QC, due to immunoreactivity variations for the same sequence synthesized by different vendors. This technique is useful in evaluating the stability of other activation/coupling reagents besides COMU and as a metrology tool for SPPS and peptide microarrays.

Diagnostic potential of the pulsed discharged helium ionization detector (PDHID) for pathogenic Mycobacterial volatile biomarkers.

Pathogenic Mycobacteria cause diseases in animals and humans with significant economic and societal consequences. Current methods for Mycobacterial detection relies upon time- and labor-intensive techniques such as culturing or DNA analysis. Using gas chromatography and mass spectrometry, four volatile compounds (methyl phenylacetate, methyl p-anisate, methyl nicotinate and o-phenyl anisole) were recently proposed as potential biomarkers for Mycobacteria. We demonstrate for the first time the capabilities of a field-deployable, pulsed discharge helium ionization detector (PDHID) for sensing these volatiles. We determined the analytical performance of the PDHID toward these Mycobacterial volatiles. Detector performance was moderately affected over the temperature range of 150 to 350 °C. The linear dynamic range for all four analytes exceeded three orders of magnitude. The limits of detection (LOD) and quantitation (LOQ) were calculated as 150 and 450 pg respectively, for all compounds, except methyl phenylacetate (LOD and LOQ, 90 and 270 pg, respectively). Control charts revealed that the PDHID detection system was generally stable, and deviations could be traced to common causes and excluded special causes. Grob tests and ionization potential data suggest that the PDHID is capable of detecting Mycobacterial volatiles in a complex milieu such as culture headspace or breath samples from tuberculosis patients. The diagnostic potential of the PDHID is critical to our goal of a handheld, field-deployable 'sniffer' system for biological pathogens and chemical warfare agents.

Surface charge dependent nanoparticle disruption and deposition of lipid bilayer assemblies.

Electrostatic interaction plays a leading role in nanoparticle interactions with membrane architectures and can lead to effects such as nanoparticle binding and membrane disruption. In this work, the effects of nanoparticles (NPs) interacting with mixed lipid systems were investigated, indicating an ability to tune both NP binding to membranes and membrane disruption. Lipid membrane assemblies (LBAs) were created using a combination of charged, neutral, and gel-phase lipids. Depending on the lipid composition, nanostructured networks could be observed using in situ atomic force microscopy representing an asymmetrical distribution of lipids that rendered varying effects on NP interaction and membrane disruption that were domain-specific. LBA charge could be localized to fluidic domains that were selectively disrupted when interacting with negatively charged Au nanoparticles or quantum dots. Disruption was observed to be related to the charge density of the membrane, with a maximum amount of disruption occurring at ∼40% positively charged lipid membrane concentration. Conversely, particle deposition was determined to begin at charged lipid concentrations greater than 40% and increased with charge density. The results demonstrate that the modulation of NP and membrane charge distribution can play a pivitol role in determining NP-induced membrane disruption and NP surface assembly.

In situ dissolution or deposition of Ytterbium (Yb) metal in microhotplate wells for a miniaturized atomic clock.

Current atomic clocks are burdened by size, weight, power and portability limitations to satisfy a broad range of potential applications. One critical need in the fabrication of a miniaturized atomic clock is small, low-power metallic sources. Exploiting the relatively high vapor pressure of ytterbium (Yb) and its dissolution in anhydrous ammonia, we report two independent techniques for depositing Yb inside a well micromachined into a microhotplate. Subsequent in situ evaporation of Yb from the microhotplate well serves as a low-power metallic source suitable for atomic clocks. The deposition and evaporation of Yb were confirmed using a variety of physicochemical techniques including quartz crystal microbalance, scanning electron microscopy, energy dispersive X-ray spectroscopy, and laser fluorescence. We also describe the fabrication of the microhotplate device, an integral component of our Yb-based miniature atomic clock. The Yb deposition/evaporation on a microhotplate well is thus useful as a low power Yb source during the fabrication of a miniaturized atomic clock, and this technique could be used for other applications requiring a vapor of a metal that has a moderate vapor pressure.

High-throughput screening of transglutaminase activity using plasmonic fluorescent nanocomposites.

We describe a high-throughput screening (HTS) assay for transglutaminase (TG) enzyme activity using plasmonic fluorescent nanocomposites. We used TG to covalently crosslink 500 µM solution of 5'-biotinamidopentylamine (BP) to N,N'-dimethylcasein (DMC) which was adsorbed onto 384-well microplates. We then bound 0.2 - 2.0 × 10(11)/mL of 10 nm gold nanoparticles-streptavidin conjugate (10 nm AuNPs-SA) to BP via biotin-streptavidin interactions. Finally, J-aggregation of cyanine 1 (25 µM) or 2 (10 µM) upon the 10 nm AuNPs elicited absorption and fluorescence signaling of TG catalysis. The cyanines could be added sequentially to elicit green (590 nm) and red (700 nm) spectral responses from the same set of reactions. Catalysis was linear (r(2) > 0.98) up to 10 min within a linear dynamic range (LDR) of 0.1 - 5 µg/mL enzyme. The multi-wavelength interrogation offered fast results (< 5 min), sensitivity (limit of detection, LOD of 5 ng or 64 fmol TG) and intermediate precision (relative standard deviation, RSD of < 20% over 42 days). Plasmonic fluorescent nanocomposites offer new ways of interrogating biomolecules in HTS format.

Plasmonic fluorescent nanocomposites of cyanines self-assembled upon gold nanoparticle scaffolds.

Plasmonic fluorescent nanocomposites are difficult to prepare due to strong quenching effects on fluorophores in the vicinity of noble metal nanoparticles such as gold (AuNPs). We successfully prepared plasmonic fluorescent nanocomposites of two cyanines (1 and 2) aggregating upon 2 - 40 nm AuNPs or streptavidin-conjugated 10 nm AuNPs. We used high throughput screening (HTS) for the first time to characterize the spectral properties, aggregation kinetics, aggregation density and photostability of the nanocomposites. Fluorescence from nanocomposites declined inversely with AuNPs size: 40 nm ≥ 20 nm > 10 nm > 5 nm > 2 nm. Sensitivity (limit of detection, LOD, 10(5) - 10(11) AuNPs/mL), brightness of the nanocomposites and surface coverage of AuNPs by cyanine aggregates were all influenced by five factors: 1) AuNPs size; 2) cyanine type (1 or 2); 3) aggregate density; 4) distance between aggregates and AuNPs surface; and 5) streptavidin protein conjugation to AuNPs. We propose a model for plasmonic fluorescent nanocomposites based on these observations. Our plasmonic fluorescent nanocomposites have applications in chemical and biological assays.

Invited article: A materials investigation of a phase-change micro-valve for greenhouse gas collection and other potential applications.

The deleterious consequences of climate change are well documented. Future climate treaties might mandate greenhouse gas (GHG) emissions measurement from signatories in order to verify compliance. The acquisition of atmospheric chemistry would benefit from low cost, small size/weight/power of microsystems. In this paper, we investigated several key materials science aspects of a phase-change microvalve (PCµV) technology with low power/size/weight/cost for ubiquitous GHG sampling. The novel design, based on phase-change material low-melting-point eutectic metal alloys (indium-bismuth, InBi and tin-lead, SnPb), could be actuated at temperatures as low as 72 °C. Valve manufacturing was based on standard thick and thin-film processes and solder technologies that are commonly used in industry, enabling low-cost, high-volume fabrication. Aging studies showed that it was feasible to batch fabricate the PCµVs and store them for future use, especially in the case of SnPb alloys. Hermetic sealing of the valve prototypes was demonstrated through helium leak testing, and Mil spec leak rates less than 1 × 10(-9) atm cm(3)/s were achieved. This confirms that the sample capture and analysis interval can be greatly expanded, easing the logistical burdens of ubiquitous GHG monitoring. Highly conservative and hypothetical CO(2) bias due to valve actuation at altitude in 1 cm(3) microsamplers would be significantly below 1.0 and 2.2 ppmv for heat-treated InBi and SnPb solders, respectively. The CO(2) bias from the PCµV scales well, as a doubling of sampler volume halved the bias. We estimated the shelf life of the SnPb PCµVs to be at least 2.8 years. These efforts will enable the development of low cost, low dead volume, small size/weight microsystems for monitoring GHGs and volatile organic compounds.

A monolithically-integrated µGC chemical sensor system.

Gas chromatography (GC) is used for organic and inorganic gas detection with a range of applications including screening for chemical warfare agents (CWA), breath analysis for diagnostics or law enforcement purposes, and air pollutants/indoor air quality monitoring of homes and commercial buildings. A field-portable, light weight, low power, rapid response, micro-gas chromatography (µGC) system is essential for such applications. We describe the design, fabrication and packaging of µGC on monolithically-integrated Si dies, comprised of a preconcentrator (PC), µGC column, detector and coatings for each of these components. An important feature of our system is that the same mechanical micro resonator design is used for the PC and detector. We demonstrate system performance by detecting four different CWA simulants within 2 min. We present theoretical analyses for cost/power comparisons of monolithic versus hybrid µGC systems. We discuss thermal isolation in monolithic systems to improve overall performance. Our monolithically-integrated µGC, relative to its hybrid cousin, will afford equal or slightly lower cost, a footprint that is 1/2 to 1/3 the size and an improved resolution of 4 to 25%.

Lipid bilayer templated gold nanoparticles nanoring formation using zirconium ion coordination chemistry.

We used positively charged lipids to prepare lipid bilayer assemblies (LBAs) upon which we assembled negatively charged gold nanoparticles (AuNPs). Treatment of the assembly with zirconium chloride resulted in the formation of nanorings of the diameters inversely related to the zirconium ion concentration. The nanorings were attributed to the zirconium ion coordinated AuNPs formed during the lipid bilayer budding process promoted by the acid effect of zirconium chloride. Nanoring organization was also dependent on the fluidity of lipid bilayers, an indication of LBA-assisted nanomaterials organization. We suggest that such bioorganic-inorganic hybrid assemblies coupled to unique topological and morphological variations might be useful as stimuli-responsive sensors or storage compartments for proteins or drugs.

Supramolecular self-assembling cyanine as an alternative to ethidium bromide displacement in DNA-drug model interactions during high throughput screening.

Supramolecular self-assembling cyanine and spermine binding to genomic DNA was a model for DNA-drug interactions during high throughput screening. Spermine competitively inhibited the self-assembly of cyanine upon DNA scaffolds as signaled by decreased fluorescence from the DNA-cyanine J-aggregate. The sequence of DNA exposure to cyanine or spermine was critical in determining the magnitude of inhibition. Methanol potentiated spermine inhibition by >10-fold. The IC(50) and association constant (K(a)) in 16% methanol were 0.35 +/- 0.03 microM and 2.86 x 10(6) M(-1) respectively, relative to 3.97 +/- 0.47 microM and 0.25 x 10(6) M(-1) respectively, in buffer. Increasing concentrations of cyanine overcame spermine inhibition, demonstrating the reversibility of DNA-drug interactions. LambdaDNA interacted similarly with spermine and cyanine, confirming system flexibility. The model drug, dye and methanol effects are discussed in detail. Cyanine might be a safer alternative to the mutagenic ethidium bromide for investigating DNA-drug interactions.

Label-free and real-time sequence specific DNA detection based on supramolecular self-assembly.

A new label-free, optical method was developed to detect sequence-specific DNA based on supramolecular self-assembly. A cationic phenylene ethynylene oligomer with two pairs of positively charged side chains (OPE-2) can form a J-dimer or J-aggregate with negatively charged DNA by a combination of electrostatic and hydrophobic interactions. At microM concentrations of dsDNA (number of bases in ssDNA ranges from 8 to 32), the optimum supramolecular self-assembly occurs between OPE-2 and dsDNA and is characterized by a new absorption peak emerging at 418 nm and an increase in fluorescence intensity (about 4.5-fold for dsDNA(1)). In contrast, the self-assembled complexes between OPE-2 and ssDNA are less readily formed under the same conditions. Interestingly, the induced circular dichroism (CD) signal for OPE-2/ssDNA is quite strong, likely owing to the self-assembly onto ssDNA simultaneously templating helix formation. In contrast, the induced CD signal for OPE-2/dsDNA is weak, likely because the dsDNA is in a double helix conformation, and OPE-2 associated with the dsDNA should be outside of the helix. In fact, there is a steady decrease in the induced CD signal for ssDNA with the addition of equimolar complementary ssDNA over time that allows the monitoring of DNA hybridization in real time. Introduction of mismatched bases into the target DNA sequence prevents the full hybridization between ssDNA and the target DNA. For these cases, the decrease in the induced CD signals occurs more slowly and to a lesser extent, as some of the unhybridized portions may remain in helical association with OPE-2. In view of these observed signal changes, sequence specific DNA and single nucleotide mismatch can be detected in a very simple and sensitive manner without any modification of the DNA.