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BACKGROUND Clozapine is the most effective antipsychotic for treatment-resistant schizophrenia. Although serum clozapine levels can help guide treatment, they are underutilized owing to requirements for frequent venous blood draws and lack of immediate results. METHODS Clozapine levels measured with a novel immunoassay technology (which enables point-of-care development) were compared with those measured by standard liquid chromatography/tandem mass spectrometry (LC-MS/MS). Frozen serum aliquots of 117 samples (N = 48 patients with schizophrenia on clozapine; N = 24 patients with schizophrenia not on clozapine; N = 45 healthy controls) were sent to a national reference laboratory (NRL) for clozapine level determination by LC-MS/MS, and matching samples were subjected to novel immunoassay (3 runs). At a later date, another frozen aliquot from the same date was sent to the NRL for repeat testing. RESULTS The NRL obtained 18 false-positive clozapine results (mean 42.39 ± 32.06, range 21-159 ng/mL) in participants not on clozapine (N = 3) and healthy controls (N = 15). The immunoassay showed no false-positive clozapine results. The clozapine levels were correlated between both assays (r = 0.84, P < 0.0001), despite 16% higher clozapine levels with immunoassay (482.08 ± 270.88 ng/mL immunoassay, 414.98 ± 186.29 ng/mL LC-MS/MS [P = 0.03]). Agreement analysis using concordance correlation coefficient (CCC) for LC-MS/MS of the 2 aliquots yielded CCC = 0.869; 95% confidence interval = 0.690-0.970, whereas higher agreement results were observed for the 3 runs of immunoassay (CCC = 0.99; 95% confidence interval = 0.979-0.997). CONCLUSIONS The lack of false positives observed with immunoassay, higher repeat performance agreement, and good correlation with LC-MS/MS may indicate the more robust performance of immunoassay than that of LC-MS/MS clozapine-level determination.

Quantitative real-time PCR (qPCR) has been the standard for nucleic acid quantification as it has a large dynamic range and good sensitivity. Digital PCR is rapidly supplanting qPCR in many applications as it provides excellent quantitative precision. However, both techniques require extensive sample preparation, and highly multiplexed assays that quantify multiple targets can be difficult to design and optimize. Here we describe a new nucleic acid quantification method that we call Spatially Isolated Reactions in a Complex Array (SIRCA), a highly parallel nucleic acid preparation, amplification, and detection approach that uses superparamagnetic microbeads in an array of thousands of 100 fL microwells to simplify sample purification and reduce reagent dispensing steps. Primers, attached to superparamagnetic microbeads through a thermo-labile bond, capture and separate target sequences from the sample. The microbeads are then magnetically loaded into a microwell array such that wells predominately contain a single bead. Master mix, lacking primers, is added before sealing the reaction wells with hydrophobic oil. Thermocycling releases the primer pair from the beads during PCR amplification. At low target concentrations, most beads capture, on average, less than one target molecule, and precise, digital PCR quantification can be derived from the percentage of positive reactions. At higher concentrations, qPCR signal is used to determine the average number of target molecules per reaction, significantly extending the dynamic range beyond the digital saturation point. We demonstrate that SIRCA can quantify DNA and RNA targets using thousands of parallel reactions, achieving attomolar limits of detection and a linear dynamic range of 105. The work reported here is a first step towards multiplexed SIRCA assays.

BACKGROUND: Clozapine is an effective antipsychotic for treatment-resistant schizophrenia for which serum levels could guide therapy decisions. It is underutilized because it requires venous draws and several-day determination time with high performance liquid chromatography-tandem mass spectrometry (LC/MS-MS). This project evaluates if clozapine measured with a novel immunoassay technology, which could be developed into a fingerstick test, correlate with LC/MS-MS. This project also assesses the impact of demographic and clinical variables on assay results. METHODS: 117 serum samples (N=48 with schizophrenia on clozapine, N=24 with schizophrenia not on clozapine and N=45 healthy controls) were included. One aliquot was sent to a national reference laboratory (NRL) for determination by LC/MS-MS and another sent to Saladax for immunoassay (MyCare® Psychiatry Clozapine Assay Kit). The agreement was compared using Concordance Correlation Coefficient (CCC). Participants’ age, sex, race-ethnicity, smoking status, ascorbic acid, co-medications and complete metabolic panel were collected. Linear regression and mixed model were performed for both technologies correspondingly to examine the impact of these variables. RESULTS: Clozapine levels by the NRL had 19 false positive readings (mean 48.8 ± 14.3, range 21–138 ng/ml) in schizophrenia participants not on clozapine (N=3) and healthy controls (N=16). The immunoassay had no false positive results. A mixed effects model (with a time average of the NRL and 3 immunoassay runs), yielded a strong Pearson correlation (r=0.843, p

SHAPE chemistries exploit small electrophilic reagents that react with the 2′-hydroxyl group to interrogate RNA structure at single-nucleotide resolution. Mutational profiling (MaP) identifies modified residues based on the ability of reverse transcriptase to misread a SHAPE-modified nucleotide and then counting the resulting mutations by massively parallel sequencing. The SHAPE-MaP approach measures the structure of large and transcriptome-wide systems as accurately as for simple model RNAs. This protocol describes the experimental steps, implemented over three days, required to perform SHAPE probing and construct multiplexed SHAPE-MaP libraries suitable for deep sequencing. These steps include RNA folding and SHAPE structure probing, mutational profiling by reverse transcription, library construction, and sequencing. Automated processing of MaP sequencing data is accomplished using two software packages. ShapeMapper converts raw sequencing files into mutational profiles, creates SHAPE reactivity plots, and provides useful troubleshooting information, often within an hour. SuperFold uses these data to model RNA secondary structures, identify regions with well-defined structures, and visualize probable and alternative helices, often in under a day. We illustrate these algorithms with the E. coli thiamine pyrophosphate riboswitch, E. coli 16S rRNA, and HIV-1 genomic RNAs. SHAPE-MaP can be used to make nucleotide-resolution biophysical measurements of individual RNA motifs, rare components of complex RNA ensembles, and entire transcriptomes. The straightforward MaP strategy greatly expands the number, length, and complexity of analyzable RNA structures.

Currently, reliable valving on integrated microfluidic devices fabricated from rigid materials is confined to expensive and complex methods. Freeze-thaw valves (FTVs) can provide a low cost, low complexity valving mechanism, but reliable implementation of them has been greatly hindered by the lack of ice nucleation sites within the valve body's small volume. Work to date has required very low temperatures (on the order of -40 °C or colder) to induce freezing without nucleation sites, making FTVs impractical due to instrument engineering challenges. Here, we report the use of ice-nucleating proteins (INPs) to induce ice formation at relatively warm temperatures in microfluidic devices. Microfluidic channels were filled with buffers containing femtomolar INP concentrations from Pseudomonas syringae. The channels were cooled externally with simple, small-footprint Peltier thermoelectric coolers (TECs), and the times required for channel freezing (valve closure) and thawing (valve opening) were measured. Under optimized conditions in plastic chips, INPs made sub-10 s actuations possible at TEC temperatures as warm as -13 °C. Additionally, INPs were found to have no discernible inhibitory effects in model enzyme-linked immunosorbent assays or polymerase chain reactions, indicating their compatibility with microfluidic systems that incorporate these widely used bioassays. FTVs with INPs provide a much needed reliable valving scheme for rigid plastic devices with low complexity, low cost, and no moving parts on the device or instrument. The reduction in freeze time, accessible actuation temperatures, chemical compatibility, and low complexity make the implementation of compact INP-based FTV arrays practical and attractive for the control of integrated biochemical assays.

Authors: Kady-Ann Steen, Nathan A. Siegfried & Kevin M. Weeks ### Abstract SHAPE (selective 2’-hydroxyl acylation analyzed by primer extension) chemistry is a powerful approach for single-nucleotide resolution analysis of RNA secondary and tertiary structure. SHAPE uses hydroxyl-selective electrophiles to react with and form covalent adducts at the ribose 2’-hydroxyl group. The most useful and robust reagent for routine SHAPE experiments is 1-methyl-7-nitroisatoic anhydride (1M7). This protocol describes a straightforward synthesis of 1M7 from commercially available precursors. The synthesis of 1-methyl-7-nitroisatoic anhydride was originally described by Mortimer et al. . The reaction scheme is:   *Amounts of each starting material can be varied using the above Table*. ### Reagents 1. 4-nitroisatoic anhydride (4NIA) (AstaTech, cat. no. 69441) - Sodium hydride (NaH) – 60% dispersion in mineral oil (Sigma-Aldrich, cat. no. 452912) - Methyl iodide (MeI) (Sigma-Aldrich, cat. no. 289566) - Anhydrous dimethylfluoride (DMF) (Sigma-Aldrich, cat. no. 227056) - HCl (Fisher Scientific, cat. no. A144) - H2O - Ether (Fisher Scientific, cat. no. E138) **REAGENT SETUP** - H2O and HCl should be ice cold before use. ### Equipment 1. Round bottom flasks (250 and 500 mL) - Bunsen burner - N2 tank and gas line - Teflon-coated stirring bar - Rubber septa - Disposable syringes (1 mL, 20 mL, 60 mL) - Disposable needles (18 gauge) - Stirring plate - Buchner funnel - Vacuum rotary evaporator - Watch glass - Oven maintained at 90 °C **EQUIPMENT SETUP** - All glassware used in the described reactions are washed with soap and water, rinsed with water, rinsed with acetone, and flame-dried over Bunsen burner. Rubber septa are immediately placed over flasks, flasks are flushed with N2, and allowed to cool. ### Procedure 1. Dissolve 4NIA in 60 mL DMF in a 250 mL flame-dried round bottom flask under N2. - In a separate 500 mL flame-dried round bottom flask under N2, make a slurry of NaH in 15 mL DMF and stir. - Slowly add 4NIA solution dropwise to slurry of NaH in DMF. Stir for 5 minutes. - Slowly add MeI dropwise and then stir at room temperature for 4 hours. - Pour reaction into 100 mL *ice cold* 1N HCl. - Filter resulting bright orange precipitate by vacuum filtration in a Buchner funnel. - Rinse precipitate twice with *ice cold* water, followed by 3 rinses with ether. - Dry overnight in oven on watch glass. ### Anticipated Results **Analytical data** Yield ~80%; bright orange powdery solid. 1H NMR [400 MHz, CO(CD3)2]: σ 3.69 (s, 3H, -NCH3-), 8.12 (dd, J=8.8 Hz, 2 Hz, 1 H, ArH), 8.2 (d, J=2hz, 1h, ArH), 8.34 (d, J=8.4 Hz, 1 H, ArH). ### Associated Publications **Selective 2′-hydroxyl acylation analyzed by protection from exoribonuclease (RNase-detected SHAPE) for direct analysis of covalent adducts and of nucleotide flexibility in RNA**. Kady-Ann Steen, Nathan A Siegfried, and Kevin M Weeks. *Nature Protocols* 6 (11) 1683 - 1694 [doi:10.1038/nprot.2011.373](http://dx.doi.org/10.1038/nprot.2011.373) ### Author information **Kady-Ann Steen, Nathan A. Siegfried & Kevin M. Weeks**, Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599-3290 Correspondence to: Kevin M. Weeks (weeks@unc.edu) *Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/2177) (2011) doi:10.1038/protex.2011.255. Originally published online 3 November 2011*.