Chiral self-assembly of fullerene clusters on CT-DNA templates.

Herein we discuss the differential interaction of three monosubstituted fullerene derivatives possessing pyridinium, aniline or phenothiazine end groups (F-Py, F-An and F-PTz, respectively) with calf thymus DNA (CT-DNA), probed via spectroscopic and imaging techniques. The pyridinium derivative, F-Py becomes molecularly dissolved in 10% DMSO-PBS and interacts with CT-DNA via groove binding and electrostatic interactions, leading to the initial condensation of CT-DNA into micrometer sized aggregates and subsequent precipitation. On the other hand, the aniline derivative F-An, which is reported to form nanoclusters of 3-5 nm size, interacts with DNA through ordered, chiral assemblies on the CT-DNA template, thus perturbing the highly networked structure of CT-DNA to form nanonetworks, which eventually transform into condensed aggregates. The binding interactions between CT-DNA and F-An nanoclusters were established via UV-Vis, AFM and TEM analysis, and the chiral nature of the fullerene nanocluster assemblies on CT-DNA was confirmed by the presence of induced circular dichroism that was exhibited around the 250-370 nm region, corresponding to F-An nanocluster absorption. In contrast, the phenothiazine derivative F-PTz, which forms larger nanoclusters of ∼70 nm size in 10% DMSO-PBS, exhibited only weak interactions with CT-DNA without affecting its network structure. These results demonstrate the role of the hydrophobic-hydrophilic balance in the design of DNA interacting fullerene derivatives by controlling their cluster size and interactions with CT-DNA, and are significant in applications such as DNA condensation, gene delivery and dimension controlled nanomaterial fabrication.

pH-Responsive Fluorescence Enhancement in Graphene Oxide-Naphthalimide Nanoconjugates: A Fluorescence Turn-On Sensor for Acetylcholine.

A pH-sensitive, fluorescence "turn-on" sensor based on a graphene oxide-naphthalimide (GO-NI) nanoconjugate for the detection of acetylcholine (ACh) by monitoring the enzymatic activity of acetylcholinesterase (AChE) in aqueous solution is reported. These nanoconjugates were synthesized by covalently anchoring picolyl-substituted NI derivatives on the GO/reduced GO surface through a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide coupling strategy, and the morphological and photophysical properties were studied in detail. Synergistic effects of π-π interactions between GO and the NI chromophore, and efficient photoinduced electron- and energy-transfer processes, were responsible for the strong quenching of fluorescence of these nanoconjugates, which were perturbed under acidic pH conditions, leading to significant enhancement of fluorescence emission. This nanoconjugate was successfully employed for the efficient sensing of pH changes caused by the enzymatic activity of AChE, thereby demonstrating its utility as a fluorescence turn-on sensor for ACh in the neurophysiological range.

Fullerene Cluster Assisted Self-Assembly of Short DNA Strands into Semiconducting Nanowires.

Programmable, hierarchical assembly of DNA nanostructures with precise organisation of functional components have been demonstrated previously with tiled assembly and DNA origami. However, building organised nanostructures with random oligonucleotide strands remains as an elusive problem. Herein, a simple and general strategy, in which nanoclusters of a fullerene derivative act as stapler motifs in bringing ordered nanoscale assembly of short oligonucleotide duplexes into micrometre-sized nanowires, is described. In this approach, the fullerene derivative, by virtue of its amphiphilic structure and unique hydrophobic-hydrophilic balance, pre-assembles to form 3-5 nm sized clusters in a mixture of DMSO-phosphate buffer, which further assists the assembly of DNA strands. The optimum cluster size, availability of DNA anchoring motifs and the nature of the DNA strands controls the structure of these nanomaterials. Furthermore, horizontal conductivity measurements through conductive AFM confirmed the charge transport properties of these nanowires. The current strategy could be employed to organise random DNA duplexes and tiles into functional nanostructures, and hence, open up new avenues in DNA nanotechnology.

Carbazole-Linked Near-Infrared Aza-BODIPY Dyes as Triplet Sensitizers and Photoacoustic Contrast Agents for Deep-Tissue Imaging.

Four new N-ethylcarbazole-linked aza-boron-dipyrromethene (aza-BODIPY) dyes (8 a,b and 9 a,b) were synthesized and characterized. The presence of the N-ethylcarbazole moiety shifts their absorption and fluorescence spectra to the near-infrared region, λ≈650-730 nm, of the electromagnetic spectrum. These dyes possess strong molar absorptivity in the range of 3-4×104 m-1 cm-1 with low fluorescence quantum yields. The triplet excited state and singlet oxygen generation of these dyes were enhanced upon iodination at the core position. The core-iodinated dyes 9 a,b showed excellent triplet quantum yields of about 90 and 75 %, with singlet oxygen generation efficiency of about 70 and 60 % relative to that of the parent dyes. Derivatives 8 a,b showed dual absorption profiles, in contrast to dyes 9 a,b, which had the characteristic absorption band of aza-BODIPY dyes. DFT calculations revealed that the electron density was spread over the iodine and dipyrromethene plane of 9 a,b, whereas in 8 a,b the electron density was distributed on the carbazole group and dipyrromethene plane of aza-BODIPY. The uniqueness of these aza-BODIPY systems is that they exhibit efficient triplet-state quantum yields, high singlet oxygen generation yields, and good photostability. Furthermore, the photoacoustic (PA) characteristics of these aza-BODIPY dyes was explored, and efficient PA signals for 8 a were observed relative to blood serum with in vitro deep-tissue imaging, thereby confirming its use as a promising PA contrast agent.

Simultaneous binding of a cyclophane and classical intercalators to DNA: observation of FRET-mediated white light emission.

DNA-assisted Förster resonance energy transfer (FRET) between an anthracene-based cyclophane (CP) and mono- and bis-intercalators such as propidium iodide (PI) and ethidium homodimer-1 (EHD), respectively, has been studied using various photophysical and biophysical techniques. The cyclophane and PI exhibited simultaneous binding to DNA at all concentrations studied and showed DNA-assisted FRET from the excimer of cyclophane with a FRET efficiency of ca. 71%. On the other hand, the bis-intercalator EHD, only at lower concentrations (<3 µM), can act as an acceptor for the energy transfer process with a lower efficiency of ca. 44%. At higher concentrations (>15 µM), EHD, on account of its higher binding affinity, displaces cyclophane from the DNA scaffold. Employing the ternary system comprising of the cyclophane, DNA and PI and fine-tuning the concentrations of the components in a molar ratio of 1 : 0.75 : 0.05 (CP : DNA : PI) we have demonstrated white light emission with CIE coordinates (0.35, 0.37).

A Pyridinium fluorophore for the detection of zinc ions under autophagy conditions.

Molecular probes for sensing and imaging of various analytes and biological specimens are of great importance in clinical diagnostics, therapy, and disease management. Since the cellular concentration of free Zn2+ varies from nanomolar to micromolar range during cellular processes and the high affinity Zn2+ imaging probes tend to saturate at lower concentrations of free Zn2+, fluorescence based probes with moderate binding affinity are desirable in distinguishing the occurrence of higher zinc concentrations in the cells. Herein, we report a new, pentacyclic pyridinium based probe, PYD-PA, having a pendant N,N-di(pyridin-2-ylmethyl)amine (DPA) for Zn2+ detection in the cellular environment. The designed probe is soluble in water and serves as a mitochondria targeting unit, whereas the pendent DPA acts as the coordination site for Zn2+. PYD-PA displayed a threefold enhancement in fluorescence intensity upon Zn2+ binding with a 1:1 binding stoichiometry. Further, the probe showed a selective response to Zn2+ over other biologically relevant metal ions with a moderate binding affinity (Ka = 6.29 × 104 M-1), good photostability, pH insensitivity, and low cytotoxicity. The demonstration of bioimaging in SK-BR-3 breast cancer cell lines confirmed the intracellular Zn ion sensing ability of the probe. The probe was successfully applied for real time monitoring of the fluctuation of intracellular free zinc ions during autophagy conditions, demonstrating its potential for cellular imaging of Zn2+ at higher intracellular concentrations.

Oxidative thymine mutation in DNA: water-wire-mediated proton-coupled electron transfer.

One-electron oxidation of A/T-rich DNA leads to mutations at thymine. Experimental investigation of DNA containing methyl-deuterated thymine reveals a large isotope effect establishing that cleavage of this carbon-hydrogen bond is involved in the rate-determining step of the reaction. First-principles quantum calculations reveal that the radical cation (electron hole) generated by DNA oxidation, initially located on adenines, localizes on thymine as the proton is lost from the methyl group, demonstrating the role of proton-coupled electron transfer (PCET) in thymine oxidation. Proton transport by structural diffusion along a segmented "water-wire" culminates in proton solvation in the hydration environment, serving as an entropic reservoir that inhibits reversal of the PCET process. These findings provide insight into mutations in A/T-rich DNA such as replication fork stalling that is implicated in early stage carcinogenesis.

Exploring a Mitochondria Targeting, Dinuclear Cyclometalated Iridium (III) Complex for Image-Guided Photodynamic Therapy in Triple-Negative Breast Cancer Cells.

Photodynamic therapy (PDT) has emerged as an efficient and noninvasive treatment approach utilizing laser-triggered photosensitizers for combating cancer. Within this rapidly advancing field, iridium-based photosensitizers with their dual functionality as both imaging probes and PDT agents exhibit a potential for precise and targeted therapeutic interventions. However, most reported classes of Ir(III)-based photosensitizers comprise mononuclear iridium(III), with very few examples of dinuclear systems. Exploring the full potential of iridium-based dinuclear systems for PDT applications remains a challenge. Herein, we report a dinuclear Ir(III) complex (IRDI) along with a structurally similar monomer complex (IRMO) having 2-(2,4-difluorophenyl)pyridine and 4'-methyl-2,2'-bipyridine ligands. The comparative investigation of the mononuclear and dinuclear Ir(III) complexes showed similar absorption profiles, but the dinuclear derivative IRDI exhibited a higher photoluminescence quantum yield (Φp) of 0.70 compared to that of IRMO (Φp = 0.47). Further, IRDI showed a higher singlet oxygen generation quantum yield (Φs) of 0.49 compared to IRMO (Φs = 0.28), signifying the enhanced potential of the dinuclear derivative for image-guided photodynamic therapy. In vitro assessments indicate that IRDI shows efficient cellular uptake and significant photocytotoxicity in the triple-negative breast cancer cell line MDA-MB-231. In addition, the presence of a dual positive charge on the dinuclear system facilitates the inherent mitochondria-targeting ability without the need for a specific targeting group. Subcellular singlet oxygen generation by IRDI was confirmed using Si-DMA, and light-activated cellular apoptosis via ROS-mediated PDT was verified through various live-dead assays performed in the presence and absence of the singlet oxygen scavenger NaN3. Further, the mechanism of cell death was elucidated by an annexin V-FITC/PI flow cytometric assay and by investigating the cytochrome c release from mitochondria using Western blot analysis. Thus, the dinuclear complex designed to enhance spin-orbit coupling with minimal excitonic coupling represents a promising strategy for efficient image-guided PDT using iridium complexes.

Biocompatible Aza-BODIPY-Biotin Conjugates for Photodynamic Therapy of Cancer.

With an objective to develop efficient photosensitizers to cancerous tissues, we synthesized two novel biocompatible sensitizers based on aza-BODIPYs incorporated with heavy atoms and biotin moieties. The bioconjugates DPR2a and DPR2b exhibited a favorable absorption range (600-750 nm) with excellent triplet-state quantum yields (up to 79%) and singlet oxygen generation yields (up to 75%). In vitro photobiological investigations employing MDA-MB-231 breast cancer cell lines exhibited rapid cellular uptake, negligible dark toxicity, and high photocytotoxicity. The mechanism of cell death of these systems was predominantly due to the mitochondrial damage, leading to apoptosis mediated via the generation of singlet oxygen-triggered reactive oxygen species. The in vivo studies with the representative conjugate DPR2a employing female NOD/SCID mice models showed inhibition in tumor growth and significantly decreased tumor volume post photodynamic therapy (PDT) treatment. Our results validate that both DPR2a and DPR2b with iodine incorporation exhibit favorable and superior photophysical and photobiological aspects and demonstrate thereby their potential applications in imaging and PDT of cancer.

Oxidatively generated damage to DNA at 5-methylcytosine mispairs.

Oxidatively generated damage to DNA has been implicated as causing mutations that lead to aging and disease. The one-electron oxidation of normal DNA leads to formation of a nucleobase radical cation that hops through the DNA until it is trapped irreversibly, primarily by reaction at guanine. It has been observed that 5-methylcytosine (C(m)) is a mutational "hot-spot". However, C(m) in a Watson-Crick base pair with G is not especially susceptible to oxidatively induced damage. Radical cation hopping is inhibited in duplexes that contain C-A or C-T mispairs, but no reaction is detected at cytosine. In contrast, we find that the one-electron oxidation of DNA that contains C(m)-A or C(m)-T mispairs results primarily in reaction at C(m) even in the presence of GG steps. The reaction at C(m) is attributed to proton coupled electron transfer, which provides a relatively low activation barrier path for reaction at 5-methylcytosine. This enhanced reactivity of C(m) in mispairs may contribute to the formation of mutational hot spots at C(m).

DNA condensation and formation of ultrathin nanosheets via DNA assisted self-assembly of an amphiphilic fullerene derivative.

DNA nanotechnology propose various assembly strategies to develop novel functional nanostructures utilizing unique interactions of DNA with small molecules, nanoparticles, polymers, and other biomolecules. Although, well defined nanostructures of DNA and amphiphilic small molecules were achieved through hybridization of covalently modified DNA, attaining precise organization of functional moieties through non-covalent interactions remain as a challenging task. Herein, we report mutually assisted assembly of an amphiphilic fullerene derivative and various DNA structures through non-covalent interactions, which leads to initial DNA condensation and subsequent assembly yielding ordered fullerene-DNA nanosheets. The molecular design of the cationic, amphiphilic fullerene derivative (FPy) ensures molecular solubility in the 10% DMSO-PBS buffer system and facile interactions with DNA through groove binding and electrostatic interactions of fullerene moiety and positively charged pyridinium moiety, respectively. The formation of FPy/DNA nanostructures were thoroughly investigated in the presence of λ-DNA, pBR322 plasmid DNA, and single and double stranded 20-mer oligonucleotides using UV-visible spectroscopy, AFM and TEM analysis. λ-DNA and pBR322 plasmid DNA readily condense in presence of FPy leading to micrometer sized few layer nanosheets with significant crystallinity due to ordered arrangement of fullerenes. Similarly, single and double stranded 20-mer oligonucleotides also interact efficiently with FPy and form highly crystalline nanosheets, signifying the role of electrostatic interaction and subsequent charge neutralization in the condensation triggered assembly. However, there is significant differences in the crystallinity and ordered arrangements of fullerenes between these two cases, where longer DNA form condensed structures and less ordered nanosheets while short oligonucleotides lead to more ordered and highly crystalline nanosheets, which could be attributed to the differential DNA condensation. Finally, we have demonstrated the addressability of the assembly using a cyanine modified single strand DNA, which also forms highly crystalline nanosheets and exhibit efficient quenching of the cyanine fluorescence upon self-assembly. These results open up new prospects in the development of functional DNA nanostructures through non-covalent interactions and hence have potential applications in the context of DNA nanotechnology.

A non-invasive ultrasensitive diagnostic approach for COVID-19 infection using salivary label-free SERS fingerprinting and artificial intelligence.

Clinical diagnostics for SARS-CoV-2 infection usually comprises the sampling of throat or nasopharyngeal swabs that are invasive and create patient discomfort. Hence, saliva is attempted as a sample of choice for the management of COVID-19 outbreaks that cripples the global healthcare system. Although limited by the risk of eliciting false-negative and positive results, tedious test procedures, requirement of specialized laboratories, and expensive reagents, nucleic acid-based tests remain the gold standard for COVID-19 diagnostics. However, genetic diversity of the virus due to rapid mutations limits the efficiency of nucleic acid-based tests. Herein, we have demonstrated the simplest screening modality based on label-free surface enhanced Raman scattering (LF-SERS) for scrutinizing the SARS-CoV-2-mediated molecular-level changes of the saliva samples among healthy, COVID-19 infected and COVID-19 recovered subjects. Moreover, our LF-SERS technique enabled to differentiate the three classes of corona virus spike protein derived from SARS-CoV-2, SARS-CoV and MERS-CoV. Raman spectral data was further decoded, segregated and effectively managed with the aid of machine learning algorithms. The classification models built upon biochemical signature-based discrimination method of the COVID-19 condition from the patient saliva ensured high accuracy, specificity, and sensitivity. The trained support vector machine (SVM) classifier achieved a prediction accuracy of 95% and F1-score of 94.73%, and 95.28% for healthy and COVID-19 infected patients respectively. The current approach not only differentiate SARS-CoV-2 infection with healthy controls but also predicted a distinct fingerprint for different stages of patient recovery. Employing portable hand-held Raman spectrophotometer as the instrument and saliva as the sample of choice will guarantee a rapid and non-invasive diagnostic strategy to warrant or assure patient comfort and large-scale population screening for SARS-CoV-2 infection and monitoring the recovery process.

Oxidation of DNA: damage to nucleobases.

All organisms store the information necessary to maintain life in their DNA. Any process that damages DNA, causing a loss or corruption of that information, jeopardizes the viability of the organism. One-electron oxidation is such a process. In this Account, we address three of the central features of one-electron oxidation of DNA: (i) the migration of the radical cation away from the site of its formation; (ii) the electronic and structural factors that determine the nucleobases at which irreversible reactions most readily occur; (iii) the mechanism of reaction for nucleobase radical cations. The loss of an electron (ionization) from DNA generates an electron "hole" (a radical cation), located most often on its nucleobases, that migrates reversibly through duplex DNA by hopping until it is trapped in an irreversible chemical reaction. The particular sequence of nucleobases in a DNA oligomer determines both the efficiency of hopping and the specific location and nature of the damaging chemical reaction. In aqueous solution, DNA is a polyanion because of the negative charge carried by its phosphate groups. Counterions to the phosphate groups (typically Na(+)) play an important role in facilitating both hopping and the eventual reaction of the radical cation with H(2)O. Irreversible reaction of a radical cation with H(2)O in duplex DNA occurs preferentially at the most reactive site. In normal DNA, comprising the four common DNA nucleobases G, C, A, and T, reaction occurs most commonly at a guanine, resulting in its conversion primarily to 8-oxo-7,8-dihydroguanine (8-OxoG). Both electronic and steric effects control the outcome of this process. If the DNA oligomer does not contain a suitable guanine, then reaction of the radical cation occurs at the thymine of a TT step, primarily by a tandem process. The oxidative damage of DNA is a complex process, influenced by charge transport and reactions that are controlled by a combination of enthalpic, entropic, steric, and compositional factors. These processes occur over a broad distribution of energies, times, and spatial scales. The emergence of a complete picture of DNA oxidation will require additional exploration of the structural, kinetic, and dynamic properties of DNA, but this Account offers insight into key elements of this challenge.

DNA Condensation Triggered by the Synergistic Self-Assembly of Tetraphenylethylene-Viologen Aggregates and CT-DNA.

Development of small organic chromophores as DNA condensing agents, which explore supramolecular interactions and absorbance or fluorescence-based tracking of condensation and gene delivery processes, is in the initial stages. Herein, we report the synthesis and electrostatic/groove binding interaction-directed synergistic self-assembly of the aggregates of two viologen-functionalized tetraphenylethylene (TPE-V) molecules with CT-DNA and subsequent concentration-dependent DNA condensation process. TPE-V molecules differ in their chemical structure according to the number of viologen units. Photophysical and morphological studies have revealed the interaction of the aggregates of TPE-V in Tris buffer with CT-DNA, which transforms the fibrous network structure of CT-DNA to partially condensed beads-on-a-string-like arrangement with TPE-V aggregates as beads via electrostatic and groove binding interactions. Upon further increasing the concentration of TPE-V, the "beads-on-a-string"-type assembly of TPE-V/CT-DNA complex changes to completely condensed compact structures with 40-50 nm in diameter through the effective charge neutralization process. Enhancement in the melting temperature of CT-DNA, quenching of the fluorescence emission of ethidium bromide/CT-DNA complex, and the formation of induced CD signal in the presence of TPE-V molecules support the observed morphological changes and thereby verify the DNA condensation abilities of TPE-V molecules. Decrease in the hydrodynamic size, increase in the zeta potential value with the addition of TPE-V molecules to CT-DNA, failure of TPE-V/cucurbit(8)uril complex to condense CT-DNA, and the enhanced DNA condensation ability of TPE-V2 with two viologen units compared to TPE-V1 with a single viologen unit confirm the importance of positively charged viologen units in the DNA condensation process. Initial cytotoxicity analysis on A549 cancer and WI-38 normal cells revealed that these DNA condensing agents are non-toxic in nature and hence could be utilized in further cellular delivery studies.

Chemical-free and scalable process for the fabrication of a uniform array of liquid-gated CNTFET, evaluated by KCl electrolyte.

Biosensors based on liquid-gated carbon nanotubes field-effect transistors (LG-CNTFETs) have attracted considerable attention, as they offer high sensitivity and selectivity; quick response and label-free detection. However, their practical applications are limited due to the numerous fabrication challenges including resist-based lithography, in which after the lithography process, the resist leaves trace level contaminations over the CNTs that affect the performance of the fabricated biosensors. Here, we report the realization of LG-CNTFET devices using silicon shadow mask-based chemical-free lithography process on a 3-in. silicon wafer, yielding 21 sensor chips. Each sensor chip consists of 3 × 3 array of LG-CNTFET devices. Field emission scanning electron microscope (FESEM) and Raman mapping confirm the isolation of devices within the array chip having 9 individual devices. A reference electrode (Ag/AgCl) is used to demonstrate the uniformity of sensing performances among the fabricated LG-CNTFET devices in an array using different KCl molar solutions. The average threshold voltage (Vth) for all 9 devices varies from 0.46 to 0.19 V for 0.1 mM to 1 M KCl concentration range. This developed chemical-free process of LG-CNTFET array fabrication is simple, inexpensive, rapid having a commercial scope and thus opens a new realm of scalable realization of various biosensors.

A Cross-Linkable Electron-Transport Layer Based on a Fullerene-Benzoxazine Derivative for Inverted Polymer Solar Cells.

The synthesis, optoelectronic characterization and device properties of a cross-linkable fullerene derivative, [6,6]-phenyl-C61 -butyric benzoxazine ester (PCBB) is reported. PCBB shows all the basic photophysical and electrochemical properties of the parent compound [6,6]-phenyl-C61 -butyric methyl ester (PCBM). Thermal cross-linking of the benzoxazine moiety in PCBB resulted in the formation of cross-linked, solvent resistive adhesive films (C-PCBB). Atomic force microscopy (AFM) and optical microscopic studies showed dramatic reduction in the roughness and aggregation behaviour of P3HT-PCBM polymer blend film upon incorporation of C-PCBB interlayer. An inverted bulk heterojunction solar cell based on the configuration ITO/ZnO/C-PCBB/P3HT-PCBM/V2 O5 /Ag achieved 4.27 % power conversion efficiency (PCE) compared to the reference device ITO/ZnO/P3HT-PCBM/V2 O5 /Ag (PCE=3.28 %). This 25 % increase in the efficiency is due to the positive effects of C-PCBB on P3HT/C-PCBB and PCBM/C-PCBB heterojunctions.

Sequence programmed DNA three-way junctions for templated assembly of fluorescent silver nanoclusters.

Fluorescent silver nanoclusters (AgNCs) templated by DNA are promising label free fluorophores with excellent photostability and tunable optical properties. Most of the reported DNA-nanocluster fluorescent tags comprise of programmed strands for the cluster formation either on the edges as overhangs or as loops on the duplex strands. Herein, we report a design strategy for sequence programmed, DNA three-way junctions (DNA-3WJ), comprising of unhybridized cytosine nucleobases in the 3WJ-center, capable of binding to silver ions and stabilizing the AgNCs. The formation of AgNCs in these DNA-3WJs were confirmed by various spectroscopic and microscopic techniques. 3WJ20-C12 comprising of 12 cytosine bases in the center of the DNA-3WJ, form fluorescent nanoclusters with an emission maximum around 630 nm and 12% fluorescence quantum yield. Control DNA-3WJs with six cytosine bases in the center (3WJ20-C6) and ones without cytosine bases (3WJ20) failed to form fluorescent AgNCs confirming the requirement of central, unhybridized cytosine bases for the stabilization of the nanoclusters. Further, the duplex arms of DNA-3WJs were shown to influence the fluorescent properties of AgNCs by varying the size and stability of the cytosine-loop structure of DNA-3WJs. Metal ion interaction studies shows the selectivity of the 3WJ20-C12/AgNCs towards Hg2+ with sensitivity in the nanomolar range.