One-pot formation of fluorescent γ-lactams having an α-phosphorus ylide moiety through three-component α(δ')-Michael reactions of phosphines with an enyne and N-tosyl aldimines.

We demonstrate a straightforward synthesis of γ-lactams possessing an α-phosphorus ylide moiety from assembly of phosphines, N-tosyl aldimines and an enyne through an initial α(δ')-attack of phosphines to an enyne in up to 79% yield. The investigated multicomponent reaction tolerates a variety of triarylphosphines and electron-poor aldimines to give γ-lactams in one pot. One of the lactams, with the tri(p-tol)phosphine and 4-cyanophenyl moiety, exhibits fluorescence emission at 447 nm with a quantum yield of 0.11.

Non-metallic nanomaterials in cancer theranostics: a review of silica- and carbon-based drug delivery systems.

The rapid development in nanomaterials has brought great opportunities to cancer theranostics, which aims to combine diagnostics and therapy for cancer treatment and thereby improve the healthcare of patients. In this review we focus on the recent progress of several cancer theranostic strategies using mesoporous silica nanoparticles and carbon-based nanomaterials. Silicon and carbon are both group IV elements; they have been the most abundant and significant non-metallic substances in human life. Their intrinsic physical/chemical properties are of critical importance in the fabrication of multifunctional drug delivery systems. Responsive nanocarriers constructed using these nanomaterials have been promising in cancer-specific theranostics during the past decade. In all cases, either a controlled texture or the chemical functionalization is coupled with adaptive properties, such as pH-, light-, redox- and magnetic field- triggered responses. Several studies in cells and mice models have implied their underlying therapeutic efficacy; however, detailed and long-term in vivo clinical evaluations are certainly required to make these bench-made materials compatible in real bedside circumstances.

DNAzyme-Amplified Label-Free Biosensor for the Simple and Sensitive Detection of Pyrophosphatase.

The level of pyrophosphatase (PPase) expression has been suggested as a potential biomarker of various cancers, and its prognostic value has been evaluated in patients suffering from lung cancer, colorectal cancer, and hyperthyroidism. However, the detection of PPase usually needs specific materials that require complicated, time-consuming reactions with restricted linear range and sensitivity, limiting their application in early clinical diagnosis. Herein, we developed a DNAzyme-based biosensor for the detection of PPase. In the presence of PPase, pyrophosphate (PPi) and Cu2+ ions released from the PPi-Cu2+-PPi complex induce the cleavage of the DNAzyme and the corresponding substrate. An apurinic/apyrimidinic (AP) site was elaborately designed within substrates that could encase the fluorophore 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND). The fluorescence of ATMND was initially quenched but restored when the DNAzyme/substrate complex was hydrolyzed with the release of ATMND. In this way, the PPase activity can be estimated by detecting the increased fluorescence of the released ATMND. Under optimized conditions, the activity of PPase could be analyzed at concentrations from 0.5 to 1000 mU, with the lowest detectable concentration being 0.5 mU. This work lays a foundation for developing a DNAzyme-amplified fluorescent biosensor with a high sensitivity, a wide linear range, and single-step operation for use as an easy diagnostic for PPase analysis.

Using cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography to determine selective serotonin reuptake inhibitors.

We have employed a rapid and highly efficient on-line preconcentration method, cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography (CSEI-sweeping-MEKC), for the analysis of selective serotonin reuptake inhibitors (SSRIs) of antidepressant drugs. We monitored the effects of several of the CSEI-sweeping-MEKC parameters - including the pH, the concentrations of high-conductivity buffer (HCB), sodium dodecyl sulfate (SDS), and organic modifier, the injection length of the HCB, and the injection time of the sample - to optimize the separation process. The optimal background electrolyte was 50 mM citric acid/disodium hydrogenphosphate buffer (pH 2.2) containing 100 mM SDS and 22% isopropyl alcohol. The sensitivity enhancements of the SSRIs sertraline, fluoxetine, paroxetine, fluvoxamine, and citalopram ranged from 5.7 x 10(4) to 1.2 x 10(5); the coefficients of determination exceeded 0.9938 and the relative standard deviations of the peak heights were less than 3.2%; the detection limits ranged from 0.056 to 0.22 ng/mL. We employed the optimal conditions to analyze these five SSRIs in a plasma sample prepared using solid-phase extraction (SPE) to minimize the influence of the matrix. Although the limits of detection of the SSRIs in human plasma were higher than those in pure water, this present technique is more sensitive than other, more-conventional methods. The recovery of the SPE extraction efficiency was satisfactory (up to 89%). Our findings suggest that, under the optimal conditions, the CSEI-sweeping-MEKC method can be used successfully to determine these five SSRIs in human plasma.

Combining DNA-stabilized silver nanocluster synthesis with exonuclease III amplification allows label-free detection of coralyne.

In this paper we describe a label-free biosensor for coralyne, prepared by combining DNA-stabilized silver nanoclusters (Ag NCs) with an exonuclease III amplification strategy. An artificial DNA probe having a polyadenine (poly-A) sequence at both the 3'- and 5'-ends was used as a probe to detect coralyne. In the absence of coralyne, the probe existed in a hairpin conformation that left both its 3'- and 5'-ends free. In the presence of coralyne, two adjacent adenine (A) bases in the poly-A sequence of the probe formed an A2 unit and then coordinated with coralyne through non-Watson-Crick base pairing. The DNA probe, having captured coralyne, was subsequently digested by exonuclease III, even though the distance between the A2 units in the A2-coralyne-A2 complex would be much larger than that found in common Watson-Crick base pairing. After digestion, the DNA probe became a single-stranded DNA (ssDNA) residue and released its captured coralyne. The liberated coralyne was then coordinated by another DNA probe having the hairpin conformation; as a result, many ssDNA residues formed after digestion. Two kinds of Ag NCs having different optical utilities were obtained: one corresponding to the hairpin conformational DNA probe and the other to the ssDNA residue. The difference in fluorescence intensity at 588 nm of these two kinds of Ag NCs reflected the concentration of coralyne. The linear range (on a logarithmic scale) for detecting coralyne spanned from 5 to 1000 nM, with an estimated detection limit of 1.83 nM.

Simultaneous Quantitation of Methamphetamine, Ketamine, Opiates and their Metabolites in Urine by SPE and LC-MS-MS.

Heroin, methamphetamine and ketamine have been the most commonly abused drugs in Taiwan. The presence of these drugs and their metabolites in postmortem specimens has been routinely monitored in our laboratory mostly by gas chromatographic-mass spectrometric methods. This study aimed to evaluate a more effective approach to simultaneously quantify these analytes (i.e., amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), morphine, codeine, 6-acetylmorphine, 6-acetylcodeine, ketamine and norketamine) in postmortem urine and blood specimens by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Samples (1 mL) were extracted via solid-phase extraction, evaporated and reconstituted in the mobile phase for injection into the LC-MS-MS system. Respective deuterated analogs of these analytes were used as internal standards. Chromatographic separation was achieved by an Agilent Zorbax SB-Aq analytical column at 50°C. Mass spectrometric analysis was performed by electrospray ionization in positive-ion dynamic multiple reaction monitoring mode with optimized collision energy for respective precursor ion selected for each analyte, and the monitoring of two transition ions. Performance characteristics were assessed using drug-free samples that were fortified with 50-1,000 ng/mL of the 10 analytes. Analytical parameters evaluated and resulting data are as follows: (i) average extraction recoveries (n= 3) were better than 80%, except for MDMA (71%) and morphine (74%); (ii) inter-day and intra-day precision ranges (%CV) were 1.59-8.80% and 0.57-3.89%, respectively; (iii) calibration linearity (r2), detection limit and quantitation limit for all analytes were >0.999, 1 and 5 ng/mL, respectively; (iv) matrix effects (ion suppression) were observed for three analytes, but were satisfactorily compensated for by the deuterated internal standards adopted in the analytical protocol. This method was successfully applied to the analysis of specimens collected from unknown death cases from various district prosecutors' offices in Taiwan, and was also found helpful to understanding whether the detected opiates were derived from heroin or legal morphine/codeine-containing medications.

Fabrication of microfluidic devices using dry film photoresist for microchip capillary electrophoresis.

An inexpensive, disposable microfluidic device was fabricated from a dry film photoresist using a combination of photolithographic and hot roll lamination techniques. A microfluidic flow pattern was prefabricated in a dry film photoresist tape using traditional photolithographic methods. This tape became bonded to a poly(methyl methacrylate) (PMMA) sheet with prepouched holes when passed through a hot roll laminator. A copper working electrode and platinum decoupler was readily incorporated within this microchip. The integrated microchip device was then fixed in a laboratory-built Plexiglas holder prior to its use in microchip capillary electrophoresis. The performance of this device with amperometric detection for the separation of dopamine and catechol was examined. The separation was complete within 50 s at an applied potential of 200 V/cm. The relative standard deviations (RSD) of analyte migration times were less than 0.71%, and the theoretical plate numbers for dopamine and catechol were 3.2 x 10(4) and 4.1 x 10(4), respectively, based on a 65 mm separation channel.

Sweeping technique combined with micellar electrokinetic chromatography for the simultaneous determination of flunitrazepam and its major metabolites.

A sweeping technique, in conjunction with micellar electrokinetic chromatography, for the simultaneous determination of flunitrazepam and its major metabolites, 7-aminoflunitrazepam and N-desmethylflunitrazepam, is described. The optimized conditions for the sweeping and separation were a pH 9.5 buffer, 25mM borate, 50mM cetyltrimethylammonium bromide, 30% MeOH (v/v), and a 151-mm injection length. The calibration functions were all linear with the coefficient of determination (r(2)) exceeding 0.996 for the three target compounds. Using the sweeping procedure, the limits of detection were determined to be 13.4, 5.6, and 12.0ng/mL for flunitrazepam, 7-aminoflunitrazepam, and N-desmethylflunitrazepam, respectively, and the sensitivity enhancement for each compound was within the range of 110-200 fold. The RSDs for the retention time and the peak area were less than 4.10%. The optimized sweeping method was also used to examine a spiked urine sample. We conclude that sweeping with micellar electrokinetic chromatography has considerable potential use in clinical and forensic analyses of flunitrazepam and its metabolites.

On-line preconcentration and determination of ketamine and norketamine by micellar electrokinetic chromatography. Complementary method to gas chromatography/mass spectrometry.

We have investigated a rapid, simple, and highly efficient on-line preconcentration method using in micellar electrokinetic chromatography (MEKC) for the analysis of abused drugs. Ketamine is an anesthetic that has been abused as a hallucinogen. We applied the sample sweeping technique first to ketamine and its major metabolite, norketamine, and separated the analytes with MEKC. Several of the sweeping MEKC parameters to effect successful separations, such as the concentration of sodium dodecyl sulfate (SDS), the injection time, and the applied voltage were optimized. The improvements in the number of theoretical plates under the different separation conditions are presented clearly in a three-dimensional representation. The limits of detection were 2.8, 3.4, and 3.3 ng/mL for ketamine, norketamine, and ketamine-D(4), respectively. The enrichment factor for each compound was within the range of 540-800. Experimental results are in agreement with those of analysis conducted by gas chromatography/mass spectroscopy (GC/MS). Therefore, we believe that sweeping, combined with MEKC, represents a suitable complementary method to GC/MS for use in clinical and forensic analyses of ketamine and norketamine.

Graphene Oxide Based Nanocarrier Combined with a pH-Sensitive Tracer: A Vehicle for Concurrent pH Sensing and pH-Responsive Oligonucleotide Delivery.

We chemically tuned the oxidation status of graphene oxide (GO) and constructed a GO-based nanoplatform combined with a pH-sensitive fluorescence tracer that is designed for both pH sensing and pH-responsive drug delivery. A series of GOs oxidized to distinct degrees were examined to optimize the adsorption of the model drug, poly dT30. We determined that highly oxidized GO was a superior drug-carrier candidate in vitro when compared to GOs oxidized to lesser degrees. In the cell experiment, the synthesized pH-sensitive rhodamine dye was first applied to monitor cellular pH; under acidic conditions, protonated rhodamine fluoresces at 588 nm (λex=561 nm). When the dT30-GO nanocarrier was introduced into cells, a rhodamine-triggered competition reaction occurred, and this led to the release of the oligonucleotides and the quenching of rhodamine fluorescence by GO. Our results indicate high drug loading (FAM-dT30/GO=25/50 µg/mL) and rapid cellular uptake (<0.5 h) of the nanocarrier which can potentially be used for targeted RNAi delivery to the acidic milieu of tumors.

Toward live-cell imaging of dopamine neurotransmission with fluorescent neurotransmitter analogues.

We report a novel 'fluorescent dopamine' that possesses essential features of natural dopamine. Our method is simple and is readily extended to monoamine neurotransmitters such as L-norepinephrine, serotonin and GABA, providing a more practical approach. Because of its compatibility with sensitive fluorescent measurements, we envisage that our approach will have a broad range of applications in neural research.

Analysis of chitin oligosaccharides by capillary electrophoresis with laser-induced fluorescence.

A method, using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection for analyzing chitin oligosaccharides is described. Chitin oligosaccharides were derivatized with 9-aminopyrene-1,4,6-trisulfonate (APTS) via reductive amination at 37 degrees C for 16 h (optimized conditions). The APTS-chitin oligosaccharides were analyzed using either an acidic citric acid-phosphate buffer or an alkaline borate buffer. The effects of buffer types, buffer pH values, and buffer concentrations on the separation were examined. The analytes were successfully separated by using a pH 4.6 citric acid-phosphate within 19 min. The APTS-derivatized chitin monosaccharide (D-glucosamine) migrated first. The analytes were also completely separated by using a pH 9.0 borate buffer within 24 min. Moreover, the specificity of enzyme digestion on chitin polysaccharides using the optimized APTS labeling procedure and the CE-LIF method was demonstrated.

Optimization of the headspace solid-phase microextraction for determination of glycol ethers by orthogonal array designs.

A headspace solid-phase microextraction (HS-SPME), in conjunction with gas chromatography-flame ionization detection for use in the determination of six frequently used glycol ethers at the microg/l level is described. A 75 microm Carboxenpolydimethylsiloxane fiber was used to extract the analytes from an aqueous solution. Experimental HS-SPME parameters such as extraction temperature, extraction time, salt concentration and sample volume, were investigated and optimized by orthogonal array experimental designs. The relative standard deviations for the reproducibility of the optimized HS-SPME method varied from 1.48 to 7.59%. The correlation coefficients of the calibration curves exceeded 0.998 in the microg/l range of concentration with at least two orders of magnitude. The method detection limits for glycol ethers in deionized water were in the range of 0.26 to 3.42 microg/l. The optimized method was also applied to the analysis of glycol ethers in urine and blood samples with the method detection limits ranged from 1.74 to 23.2 microg/l.

Oligonucleotides as 'bio-solvent' for in situ extraction and functionalisation of carbon nanoparticles.

A simple yet novel one-pot approach is developed to prepare carbon nanoparticles with diameters of ∼2 nm and modified by oligonucleotides. We use single-stranded deoxyribonucleic acid (ssDNA), which serves as a unique 'bio-solvent' for carbon nanoparticle (CNP) preparation and as a target molecule for functionalisation. Proposed interactions relevant to the stabilisation of the final oligonucleotide-CNP complex include π-π stacking and π-HN bonding with sp2 carbon atoms on the CNP surface. Furthermore, oligonucleotide-enriched CNPs can be readily extracted within seconds from a crude mixture of single-walled carbon nanotubes (SWCNTs) without any need for post-synthesis chemical modification. The established CNPs are biocompatible, possess intrinsic fluorescence, and do not result in the undesirable photobleaching effect, rendering them potential candidates for in vivo biological applications.

Sensitive label-free electrochemical analysis of human IgE using an aptasensor with cDNA amplification.

In this study, we developed an ultrasensitive label-free aptamer-based electrochemical biosensor, featuring a highly specific anti-human immunoglobulin E (IgE) aptamer as a capture probe, for human IgE detection. Construction of the aptasensor began with the electrodeposition of gold nanoparticles (AuNPs) onto a graphite-based screen-printed electrode (SPE). After immobilizing the thiol-capped anti-human IgE aptamer onto the AuNPs through self-assembly, we treated the electrode with mercaptohexanol (MCH) to ensure that the remaining unoccupied surfaces of the AuNPs would not undergo nonspecific binding. We employed a designed complementary DNA featuring a guanine-rich section in its sequence (cDNA G1) as a detection probe to bind with the unbound anti-human IgE aptamer. We measured the redox current of methylene blue (MB) to determine the concentration of human IgE in the sample. When the aptamer captured human IgE, the binding of cDNA G1 to the aptamer was inhibited. Using cDNA G1 in the assay greatly amplified the redox signal of MB bound to the detection probe. Accordingly, this approach allowed the linear range (coefficient of determination: 0.996) for the analysis of human IgE to extend from 1 to 100,000pM; the limit of detection was 0.16pM. The fabricated aptasensor exhibited good selectivity toward human IgE even when human IgG, thrombin, and human serum albumin were present at 100-fold concentrations. This method should be readily applicable to the detection of other analytes, merely by replacing the anti-human IgE aptamer/cDNA G1 pair with a suitable anti-target molecule aptamer and cDNA.

A pentacyclic nitrogen-bridged thienyl-phenylene-thienyl arene for donor-acceptor copolymers: synthesis, characterization, and applications in field-effect transistors and polymer solar cells.

A pentacyclic benzodipyrrolothiophene (BDPT) unit, in which two outer thiophene rings are covalently fastened with the central phenylene ring by nitrogen bridges, was synthesized. The two pyrrole units embedded in BDPT were constructed by using one-pot palladium-catalyzed amination. The coplanar stannylated Sn-BDPT building block was copolymerized with electron-deficient thieno[3,4-c]pyrrole-4,6-dione (TPD), benzothiadiazole (BT), and dithienyl-diketopyrrolopyrrole (DPP) acceptors by Stille polymerization. The bridging nitrogen atoms make the BDPT motif highly electron-abundant and structurally coplanar, which allows for tailoring the optical and electronic properties of the resultant polymers. Strong photoinduced charge-transfer with significant band-broadening in the solid state and relatively higher oxidation potential are characteristic of the BDPT-based polymers. Poly(benzodipyrrolothiophene-alt-benzothiadiazole) (PBDPTBT) achieved the highest field-effect hole mobility of up to 0.02 cm(2) V(-1) s(-1). The photovoltaic device using the PBDPTBT/PC(71)BM blend (1:3, w/w) exhibited a V(oc) of 0.6 V, a J(sc) of 10.34 mA cm(-2), and a FF of 50%, leading to a decent PCE of 3.08%. Encouragingly, the device incorporating poly(benzodipyrrolothiophene-alt-thienopyrrolodione) (PBDPTTPD)/PC(71)BM (1:3, w/w) composite delivered a highest PCE of 3.72%. The enhanced performance arises from the lower-lying HOMO value of PBDPTTPD to yield a higher V(oc) of 0.72 V.

1-Butyl-3-methylimidazolium-based ionic liquids and an anionic surfactant: excellent background electrolyte modifiers for the analysis of benzodiazepines through capillary electrophoresis.

In this study, we found that adding 1-butyl-3-methylimidazolium-based ionic liquids (ILs) and sodium dodecyl sulfate (SDS) as modifiers in the background electrolyte (BGE) for capillary electrophoresis enhanced the separation of benzodiazepines. In particular, 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide ([BMIM][NTf2]) was the best IL additive for the separation system because its anionic moiety interacted favorably with the benzodiazepines. We added SDS because of its known effect on the separation of hydrophobic analytes. We optimized the separation conditions in terms of the concentrations of the IL, SDS, and organic solvent, the pH, and the BGE's ionic strength. The optimal BGE, containing 170 mM [BMIM][NTf2] and 10 mM SDS, provided baseline separation, high efficiency, and satisfactory peak shapes for the benzodiazepines. The separation mechanism was based on heteroassociation between the anionic moiety of the IL and the benzodiazepines, with SDS improving the resolution of the separation. The limits of detection for the seven analytes ranged from 2.74 to 4.42 microg/mL. We subjected a urine sample to off-line solid phase extraction (SPE) prior to the analysis of its benzodiazepine content. Our experimental results reveal that the combination of [BMIM][NTf2] and SDS provides adequate separation efficiency for its application to CE analyses of benzodiazepines after SPE concentration.

Separation of open-cage fullerenes using nonaqueous capillary electrophoresis.

In this study, nonaqueous capillary electrophoresis (NACE) was used to separate three open-cage fullerenes. Trifluoroacetic acid (TFA) was used as the nonaqueous background electrolyte to change the analytes' mobilities. The selectivity and separation efficiency were critically affected by the nature of the buffer system, the choice of organic solvent, and the concentrations of TFA and sodium acetate (NaOAc) in the background electrolyte. The optimized separation occurred using 200 mM TFA/20mM NaOAc in MeOH/acetonitrile (10:90, v/v), providing highly efficient baseline separation of the open-cage fullerenes within 5 min. The migration time repeatability for the three analytes was less than 1% (relative standard deviation). Thus, NACE is a rapid, useful alternative to high-performance liquid chromatography for the separation of open-cage fullerenes.

Using sweeping micellar electrokinetic chromatography to analyze Delta9-tetrahydrocannabinol and its major metabolites.

We have applied sweeping micellar electrokinetic chromatography (sweeping-MEKC) to the simultaneous determination of Delta(9)-tetrahydrocannabinol (THC) and its major metabolites, 11-hydroxy-Delta(9)-tetrahydrocannabinol (THC-OH) and 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH). We monitored the effects of several of the sweeping-MEKC parameters, including the proportion of organic modifier, the concentration of sodium dodecyl sulfate (SDS), the pH, and the sample injection volume, to optimize the separation process. The optimal buffer for the analysis of the three analytes was 25 mM citric acid/disodium hydrogenphosphate (pH 2.6) containing 40% methanol and 75 mM SDS. Under the optimized separation parameters, the enrichment factors for THC, THC-COOH, and THC-OH when using sweeping-MEKC (relative to MEKC) were 77, 139, and 200, respectively. The limits of detection (LODs) for the three compounds in standard solutions ranged from 3.87 to 15.2 ng/mL. We combined the sweeping-MEKC method with solid-phase extraction to successfully detect THC, THC-COOH, and THC-OH in human urine with acceptable repeatability. The LODs of these analytes in urine samples ranged from 17.2 to 23.3 ng/mL. Therefore, this sweeping-MEKC method is useful for determining, with high sensitivity, the amounts of THC and its metabolites in the urine of suspected THC users.

Using the cationic surfactants N-cetyl-N-methylpyrrolidinium bromide and 1-cetyl-3-methylimidazolium bromide for sweeping-micellar electrokinetic chromatography.

This paper describes a sweeping-micellar electrokinetic chromatography (sweeping-MEKC) technique for the determination of seven benzodiazepines, using, as sweeping carriers, the ionic liquid-type cationic surfactants 1-cetyl-3-methylimidazolium bromide (C(16)MIMBr) and N-cetyl-N-methylpyrrolidinium bromide (C(16)MPYB). These surfactants resemble the commonly employed cationic surfactant cetyltrimethylammonium bromide (CTAB), but they provide different separation efficiencies. We optimized the separation and sweeping conditions, including the pH, the concentrations of organic modifier and surfactant, and the sample injection volume. Adding C(16)MIMBr or C(16)MPYB to the background electrolyte enhanced the separation efficiency and detection sensitivity during the sweeping-MEKC analyses of the benzodiazepines. C(16)MIMBr enhanced the sensitivity for each benzodiazepine 31-59-fold; C(16)MPYB, 86-165-fold. In the presence of C(16)MPYB, the limits of detection for the seven analytes ranged from 4.68 to 9.75 ng/mL. We adopted the sweeping-MEKC conditions optimized for C(16)MPYB to satisfactorily analyze a human urine sample spiked with the seven benzodiazepines. To minimize the matrix effects, we subjected this urine sample to off-line solid phase extraction (SPE) prior to analysis. The recoveries of the analytes after SPE were satisfactory (ca. 77.0-88.3%). Our experimental results reveal that the cationic surfactant C(16)MPYB exhibits superior sweeping power relative to those of C(16)MIMBr and CTAB and that it can be applied in sweeping-MEKC analyses for the on-line concentrating and analyzing of benzodiazepines present in real samples at nanogram-per-milliliter concentrations.