Rapid prototyping of electrochromatography chips for improved two-photon excited fluorescence detection.

In the present study, we introduce two-photon excitation at 532 nm for label-free fluorescence detection in chip electrochromatography. Two-photon excitation at 532 nm offers a promising alternative to one-photon excitation at 266 nm, as it enables the use of economic chip materials instead of fused silica. In order to demonstrate these benefits, one-photon and two-photon induced fluorescence detection are compared in different chip layouts and materials with respect to the achievable sensitivity in the detection of polycyclic aromatic hydrocarbons (PAHs). Customized chromatography chips with cover or bottom slides of different material and thickness are produced by means of a rapid prototyping method based on liquid-phase lithography. The design of thin bottom chips (180 µm) enables the use of high-performance immersion objectives with low working distances, which allows one to exploit the full potential of two-photon excitation for a sensitive detection. The developed method is applied for label-free analysis of PAHs separated on a polymer monolith inside polymer glass sandwich chips made from fused silica or soda-lime glass. The obtained limits of detection range from 40 nM to 1.95 µM, with similar sensitivities in fused silica thin bottom chips for one-photon and two-photon excitation. In deep-UV non- or less-transparent devices two-photon excitation is mandatory for label-free detection of aromatics with high sensitivity.

Label-free fluorescence detection of aromatic compounds in chip electrophoresis applying two-photon excitation and time-correlated single-photon counting.

In this study, we introduce time-resolved fluorescence detection with two-photon excitation at 532 nm for label-free analyte determination in microchip electrophoresis. In the developed method, information about analyte fluorescence lifetimes is collected by time-correlated single-photon counting, improving reliable peak assignment in electrophoretic separations. The determined limits of detection for serotonin, propranolol, and tryptophan were 51, 37, and 280 nM, respectively, using microfluidic chips made of fused silica. Applying two-photon excitation microchip separations and label-free detection could also be performed in borosilicate glass chips demonstrating the potential for label-free fluorescence detection in non-UV-transparent devices. Microchip electrophoresis with two-photon excited fluorescence detection was then applied for analyses of active compounds in plant extracts. Harmala alkaloids present in methanolic plant extracts from Peganum harmala could be separated within seconds and detected with on-the-fly determination of fluorescence lifetimes.

Monitoring on-chip Pictet-Spengler reactions by integrated analytical separation and label-free time-resolved fluorescence.

High-throughput screening for optimal reaction conditions and the search for efficient catalysts is of eminent importance in the development of chemical processes and for expanding the spectrum of synthetic methodologies in chemistry. In this context we report a novel approach for a microfluidic chemical laboratory integrating organic synthesis, separation and time-resolved fluorescence detection on a single microchip. The feasibility of our integrated laboratory is demonstrated by monitoring the formation of tetrahydroisoquinoline derivatives by Pictet-Spengler condensation. After on-chip reaction the products and residual starting material were separated enantioselectively on the same chip. On-chip deep UV laser-induced fluorescence detection with time-correlated single photon counting was applied for compound assignment. The system was utilized to screen reaction conditions and various substrates for Pictet-Spengler reactions on-chip. Finally, the microlab was successfully applied to investigate enantioselective reactions using BINOL-based phosphoric acids as organocatalysts.

Microfluidic chips for chirality exploration.

Microfluidic chips applied to the investigation of chirality allow reaction, separation and analysis of minuscule amounts of enantiomeric molecules. Chiral chip technology is employed in fields as diverse as pharmaceutical high throughput screening and deep space exploration missions.

Label-free analysis in chip electrophoresis applying deep UV fluorescence lifetime detection.

Herein we introduce deep UV fluorescence lifetime detection in microfluidics applied for label-free detection and identification of various aromatic analytes in chip electrophoresis. For this purpose, a frequency quadrupled Nd:YAG (neodymium-doped yttrium aluminum garnet) picosecond laser at 266 nm was incorporated into an inverse fluorescence microscope setup with time-correlated single photon counting detection. This allowed recording of photon timing with sub-nanosecond precision. Thereby fluorescence decay curves are gathered on-the-fly and average lifetimes can be determined for each substance in the electropherogram. The aromatic compounds serotonin, propranolol, 3-phenoxy-1,2-propanediol and tryptophan were electrophoretically separated using a fused-silica microchip. Average lifetimes were independently determined for each compound via bi-exponential tail fitting. Time-correlated single photon counting also allows the discrimination of background fluorescence in the time domain. This results in improved signal-to-noise-ratios as demonstrated for the above model analytes. Microchip electrophoretic separations with fluorescence lifetime detection were also performed with a protein mixture containing lysozyme, trypsinogen and chymotrypsinogen emphasizing the potential for biopolymer analysis.

An integrated on-chip sirtuin assay.

A microchip-based assay to monitor the conversion of peptide substrates by human recombinant sirtuin 1 (hSIRT1) is presented. For this purpose a fused silica microchip consisting of a microfluidic separation structure with an integrated serpentine micromixer has been used. As substrate for the assay, we used a 9-fluorenylmethoxycarbonyl (Fmoc)-labeled tetrapeptide derived from the amino acid sequence of p53, a known substrate of hSIRT1. The Fmoc group at the N-terminus resulting from solid-phase peptide synthesis enabled deep UV laser-induced fluorescence detection with excitation at 266 nm. The enzymatic reaction of 0.1 U/µL hSIRT1 was carried out within the serpentine micromixer using a 400 µM solution of the peptide in buffer. In order to reduce protein adsorption, the reaction channel was dynamically coated with hydroxypropylmethyl cellulose. The substrate and the deacetylated product were separated by microchip electrophoresis on the same chip. The approach was successfully utilized to screen various SIRT inhibitors.

Excessive urinary excretion of isopropyl glucuronide after isopropanol abuse.

BACKGROUND: Ethyl glucuronide (EtG) in urine is considered a marker of alcohol consumption. We present a case of a false-positive immunological EtG screening result due to excessive isopropyl glucuronide excretion in urine of an alcohol-dependent patient with a history of industrial cleaning fluid abuse. METHODS: EtG screening was done with the Microgenics DRI EtG enzyme immunoassay on a Beckman Coulter AU680 analyzer according to the testkit instructions. Confirmatory analysis was done by LC-MS/MS for EtG, 1-propyl (syn. n-propyl), 2-propyl (syn. isopropyl), 1-butyl, 2-butyl, and tert-butyl glucuronide. Both methods were validated according to the Guidelines of the Society of Toxicological and Forensic Chemistry (GTFCh, Germany). RESULTS: EtG screening by immunoassay was positive, approx. 860mg/L or approx. 1540mg/g creatinine (forensic cut-off 0.1mg/L, clinical cut-off 0.5mg/L). LC-MS/MS confirmatory analysis was negative for EtG (<0.05mg/L; forensic cut-off 0.1mg/L), but strongly positive for 2-propyl glucuronide (approx. 1100mg/L or 2000mg/g creatinine; cut-off 0.1mg/L). 1-propyl, 1-butyl, and tert-butyl glucuronide were negative (<0.05mg/L; cut-off 0.1mg/L), 2-butyl glucuronide was 0.1mg/L (cut-off 0.1mg/L). CONCLUSION: Consumption of household and industrial chemicals with short chain aliphatic alcohols should be considered a rare but potential source of false-positive EtG immunoassay results. Glucuronides from frequently used short chain aliphatic alcohols, like 1-propanol (syn. n-propanol) and 2-propanol (syn. isopropanol) as the most important disinfectant components, should be included into EtG confirmatory analysis. This will be helpful not only for the assessment of the source for remarkable EtG immunoassay results, it can also contribute to a more specific diagnosis in cases with suspected intoxication by consumer or industrial chemical products. Excessive urinary 2-propyl glucuronide (syn. isopropyl glucuronide) concentrations should be considered a marker of isopropanol intoxication.

Cross-reaction of propyl and butyl alcohol glucuronides with an ethyl glucuronide enzyme immunoassay.

BACKGROUND: Ethyl glucuronide (EtG) in urine is considered a marker of recent alcohol consumption. Using immunoassays for EtG screening without confirmatory analysis bears a risk of getting false-positives as shown for trichloroethyl glucuronide from chloral hydrate medication and 1-propyl glucuronide from propanol-based hand disinfection. The aim of the study was to check whether glucuronides of frequently used aliphatic short chain alcohols aside from EtG and 1-propyl glucuronide can cross-react with the DRI(®) Ethyl Glucuronide Assay. METHODS: Aliquots of EtG-free urine were individually spiked with methyl ß-D-glucuronide, 1-propyl ß-D-glucuronide, 2-propyl ß-D-glucuronide, 1-butyl ß-D-glucuronide, 2-butyl ß-D-glucuronide, and tert-butyl ß-D-glucuronide. To check the response rate of the DRI(®) Ethyl Glucuronide Assay to its target analyte, EtG was also added to a native EtG-free urine sample. The spiked alcohol glucuronide concentrations (seven levels up to 10mg/L) and the DRI(®) Ethyl Glucuronide Assay results were evaluated by Passing-Bablok regression analysis. The 95% confidence interval ranges for the slope of the regression function were considered a measure of cross-reaction of the individual alcohol glucuronides with the enzyme immunoassay. RESULTS: 2-Propyl glucuronide showed a cross-reactivity of 69-84% at the 95% probability level, methyl glucuronide, 1-propyl glucuronide, and 1- and 2-butyl glucuronide of 4-9%, and tert-butyl glucuronide almost no cross-reactivity. The response rate for EtG was 87-94% at the 95% probability level. CONCLUSION: The DRI(®) Ethyl Glucuronide Assay shows cross-reaction rates with aliphatic short chain alcohol glucuronides aside from EtG which bear a risk of getting false-positives regarding ethanol consumption. Mass spectrometric detection of EtG is mandatory for confirmation of positive immunological EtG screenings.