Christopher George (Presenter)
Protea Biosciences, Inc.
Authorship: Christopher R. George, Heather Anderson, Nicholas J. Morris, Matthew J. Powell, Haddon Goodman, Trust T. Razunguzwa
Protea Biosciences Inc., 1311 Pineview Drive, Suite 501, Morgantown, WV, 26505
| Short Abstract REDIchips are comprised of nanopost arrays (NAPA), fabricated on silicon substrates to form patterned targets for high throughput LDI-MS of small molecules in MALDI instruments. The REDIchip’s NAPA targets exhibit excellent performance for quantitation and screening of pain panel drugs such as opiates/opioids, anesthetics, and cocaine metabolites in biofluids. The pain drugs are quantitated over at least four orders of magnitude dynamic range to determine appropriate cut-off values for pass/fail parameters for drug screening from a variety of human biofluids. REDIchip’s NAPA LDI-MS workflow allows for efficient and sensitive detection and quantitation of several classes of pain drugs. |
Long Abstract
Introduction
Since its introduction in the 1980s, MALDI has become the dominant Laser Desorption Ionization (LDI) method. MALDI-MS utilizes an organic matrix to promote soft ionization of molecules and it is a well-established, sensitive, and high throughput technique that is essential for the analysis of large molecules, especially proteins, peptides, oligosaccharides, and oligonucleotides. MALDI-MS has therefore been a very effective tool in genomics, proteomics, and clinical research. It has also been used for MS imaging of macromolecules from a variety of sample sources including animal tissues. The major limitation of MALDI is the use of an external matrix which is added to the sample to absorb laser energy for sample desorption and to provide protons for the ionization. The addition of this matrix can create problems, such as poor reproducibility and unwanted chemical background peaks in the low mass region. These limitations have led to the development of matrix-free tools, such as the nanopost array (NAPA) devices fabricated in this work, to provide a surface of silicon nanoposts from which samples can be desorbed by absorption of laser energy, without external matrix requirements. The NAPA devices (REDIchips) are amenable to high throughput workflows for screening and quantitation using MALDI platforms and robotic spotting. “Matrix-free” MALDI can be carried out using these devices, meaning the low m/z region of mass spectra is useful, as the chemical noise from an external matrix is eliminated. Demonstrated here is the quantitation of pain panel drugs from aqueous solutions and screening of the drugs in human biofluids.
Methods
REDIchips were fabricated using deep ultraviolet (DUV) projection lithography and deep reactive ion etching (DRIE). A silicon dioxide layer was first grown using plasma-enhanced chemical vapor deposition (PECVD) on the bare pSi wafer prior to spin coating with photoresist. This oxide layer (~100 nm) acts as a 'hard mask' to protect the Si underneath during etching. After the SiO2 layer was grown, the wafer was patterned using photoresist and exposed using DUV projection lithography. During DRIE, an initial etch removes the SiO2 not protected by the photoresist layer. After this oxide etch, a deep-Si etch was performed, yielding the desired nanopost arrays. Fluorous modification of REDIchips was performed using (heptadecafluoro-1,1,2,2-tetrahydrodecyl) trimethoxysilane, by placing 1 μL of the fluorous compound on a NAPA target and incubating at 60oC for one hour. A dilution series of the drug molecules from 1-10000 pg/µL (1-10000 ng/mL) were prepared in aqueous solution along with deuterated internal standards at 500 pg/μL (500 ng/mL) concentration. Volumes of 1 μL were spotted on the REDIchip, allowed to dry and analyzed using an AB Sciex 4800 mass spectrometer. For screening drugs in biofluid samples, the samples were fortified with 0.5, 1, 100, 500, 1000, and 10000 pg/µL level of the drugs. The biofluid samples were centrifuged and the supernatant was collected. The supernatant was then diluted to 5% and directly spotted on a REDIchip NAPA target for analysis.
Results
Most of the drug molecules showed excellent linearity over four orders of magnitude with a detection limit close to 1 pg for 1 μL volumes of spotted samples, well below the physiological levels. Accuracy and precision were determined for each drug analyzed. Each amount of sample analyzed in triplicate (3 spots) and the spot to spot reproducibility was less than 15% RSD. Accuracy values ranged between 90-110%. Once the cut-off value for each drug was determined, several concentrations of each abuse drug was tested based on the pass/fail parameters determined. The nanopost arrays can be modified with fluorous chemistries for improved detection limits from the biofluids, demonstrated by the drug screening experiments.
Conclusions
REDIchips are nanophotonic devices that can be effectively used for high throughput screening of pain panel drugs in biofluids using Laser Desorption Ionization Mass Spectrometry.
References & Acknowledgements:
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