Electrochemical Genetic Profiling of Single Cancer Cells.

Recent understandings in the development and spread of cancer have led to the realization of novel single cell analysis platforms focused on circulating tumor cells (CTCs). A simple, rapid, and inexpensive analytical platform capable of providing genetic information on these rare cells is highly desirable to support clinicians and researchers alike to either support the selection or adjustment of therapy or provide fundamental insights into cell function and cancer progression mechanisms. We report on the genetic profiling of single cancer cells, exploiting a combination of multiplex ligation-dependent probe amplification (MLPA) and electrochemical detection. Cells were isolated using laser capture and lysed, and the mRNA was extracted and transcribed into DNA. Seven markers were amplified by MLPA, which allows for the simultaneous amplification of multiple targets with a single primer pair, using MLPA probes containing unique barcode sequences. Capture probes complementary to each of these barcode sequences were immobilized on a printed circuit board (PCB) manufactured electrode array and exposed to single-stranded MLPA products and subsequently to a single stranded DNA reporter probe bearing a HRP molecule, followed by substrate addition and fast electrochemical pulse amperometric detection. We present a simple, rapid, flexible, and inexpensive approach for the simultaneous quantification of multiple breast cancer related mRNA markers, with single tumor cell sensitivity.

Medium-high resolution electrochemical genotyping of HLA-DQ2/DQ8 for detection of predisposition to coeliac disease.

Coeliac disease is a small intestinal disorder, induced by ingestion of gluten in genetically predisposed individuals. Coeliac disease has been strongly linked to human leukocyte antigens (HLA) located on chromosome 6, with almost 100 % of coeliac disease sufferers carrying either a HLA-DQ2 or HLA-DQ8 heterodimer, with the majority carrying HLA-DQ2 encoded by the DQA1*05:01/05:05, DQB1*02:01/02:02 alleles, whereas the remaining carry the HLA-DQ8 encoded by the DQA1*03:01, DQB1*03:02 alleles. In this work, we present the development of a multiplex electrochemical genosensor array of 36 electrodes, housed within a dedicated microfluidic platform and using a total of 10 sequence-specific probes for rapid medium-high resolution HLA-DQ2/DQ8 genotyping. An evaluation of the selectivity of the designed probes was carried out with the target sequences and 44 potentially interfering alleles, including single base mismatch differentiations; good selectivity was demonstrated. The performance of the electrochemical genosensor array was validated, analyzing real human samples for the presence of HLA-DQ2/DQ8 alleles, and compared with those obtained using laboratory-based HLA typing, and an excellent correlation was obtained.

PoCOsteo: generic novel platform for bone turnover marker measurement & monitoring.

Despite the increasing efforts in improving bone health assessments, current diagnostics suffer from critical shortcomings. The present article therefore describes a multiplex label-free immunosensor designed and validated for the assessment of two bone turnover markers (BTMs), namely beta isomerized C-terminal telopeptide of type I collagen (CTx) and Procollagen I Intact N-Terminal (PINP), the combination of which is needed to illustrate an accurate overview of bone health. The immunosensor was then tested outside and inside of a microsystem, with the aim of becoming compatible with a point of care system fabricated for automated assessment of these biomarkers later-on at patient side. Custom-made monoclonal antibodies were specifically designed for this purpose in order to guarantee the selectivity of the immunosensor. In the final platform, a finger prick blood sample is introduced into the microfluidic manifolds without any need for sample preparation step, making the tool suitable for near patient and outside of the central laboratory applications. The platform was exploited in 30 real blood samples with the results validated using electrochemiluminescence immunoassay. The results revealed the platform was capable of measuring the target analyte with high sensitivity and beyond the recommended clinical reference range for each biomarker (CTx: 104-1028 ng L-1 and PINP: 16-96 µg L-1, correspondingly). They also showed the platform to have a limit of detection of 15 (ng L-1) and 0.66 (µg L-1), a limit of quantification of 49 (ng L-1) and 2.21 (µg L-1), and an inter- and intra-assay coefficient of variance of 5.39-6.97% and 6.81-5.37%, for CTx and PINP respectively, which is comparable with the gold standard. The main advantage of the platform over the state-of-the art was the capability of providing the results for two markers recommended for assessing bone health within 15 minutes and without the need for skilled personnel or costly infrastructure.

Fast nucleic acid amplification for integration in point-of-care applications.

An ultrafast microfluidic PCR module (30 PCR cycles in 6 min) based on the oscillating fluid plug concept was developed. A robust amplification of native genomic DNA from whole blood samples could be achieved at operational conditions established from systematic investigations of key parameters including heat transfer and in particular flow velocities. Experimental data were augmented with results from computational fluid dynamics simulations. The reproducibility of the current system was substantially improved compared to previous concepts by integration of a closed reservoir instead of utilizing a vented channel end at ambient pressure rendering the devised module suitable for integration into complex sample-to-answer analysis platforms such as point-of-care applications.

Automated microsystem for electrochemical detection of cancer markers.

The development of a fully automated microsystem housing an amperometric immunosensor is presented. The microfluidic cell integrates reagent storage and electrochemical immunodetection and was applied for the detection of breast cancer markers. The main advantage of this system is that no external fluidic storage is required and the instrumental setup is thus greatly simplified. The fluidics of the microsystem is computer controlled and requires minimal end-user intervention. The analytical performance of the device was compared with a manually driven system and applied for the amperometric detection of the carcinoembryonic antigen (CEA) and cancer antigen 15-3 (CA15-3). This automation methodology greatly improves the analytical performance of the immunosensor in terms of accuracy and reproducibility as evidenced by a reduction of LOD observed for CEA and CA15-3 with respect to a manually driven system. Finally, the automated microsystem was applied for the analysis of real patient serum samples, demonstrating excellent correlation with a commercial ELISA.

The utility of high-flow oxygen during emergency department procedural sedation and analgesia with propofol: a randomized, controlled trial.

STUDY OBJECTIVE: We determine whether high-flow oxygen reduces the incidence of hypoxia by 20% in adults receiving propofol for emergency department (ED) sedation compared with room air. METHODS: We randomized adults to receive 100% oxygen or compressed air at 15 L/minute by nonrebreather mask for 5 minutes before and during propofol procedural sedation. We administered 1.0 mg/kg of propofol, followed by 0.5 mg/kg boluses until the patient was adequately sedated. Physicians and patients were blinded to the gas used. Hypoxia was defined a priori as an oxygen saturation less than 93%; respiratory depression was defined as an end tidal CO(2) greater than 50 mm Hg, a 10% absolute change from baseline, or loss of waveform. RESULTS: We noted significantly less hypoxia in the 59 patients receiving high-flow oxygen compared with the 58 receiving compressed air (19% versus 41%; P=.007; difference 23%; 95% confidence interval 6% to 38%). Respiratory depression was similar between groups (51% versus 48%; difference 2%; 95% confidence interval -15% to 22%). We observed 2 adverse events in the high-flow group (1 hypotension, 1 bradycardia) and 2 in the compressed air group (1 assisted ventilation, 1 hypotension). CONCLUSION: High-flow oxygen reduces the frequency of hypoxia during ED propofol sedation in adults.

Amperometric immunosensor for carcinoembryonic antigen in colon cancer samples based on monolayers of dendritic bipodal scaffolds.

Detection of proteins that signal the presence or recurrence of cancer is a powerful therapeutic tool for effective early diagnosis and treatment. Carcinoembryonic antigen (CEA) has been extensively studied as a tumor marker in clinical diagnosis. We report on the development of an amperometric biosensor for the detection of CEA based on the immobilization of anti-CEA monoclonal antibody on a novel class of bipodal thiolated self-assembled monolayers containing reactive N-hydroxysuccinimide (NHS) ester end groups. The current variations showed a linear relationship with the concentration of CEA over the range of 0-200 ng/mL with a sensitivity of 3.8 nA x mL x ng(-1) and a detection limit of 0.2 ng/mL, which is well below the commonly accepted concentration threshold (5 ng/mL) used in clinical diagnosis. Real time and accelerated stability studies of the reporter antibody under various storage conditions demonstrated that the enzymatic activity and antibody affinity of the conjugate is retained for long periods of time in commercial stabilizing buffers such as StabilGuard Biomolecule Stabilizer, and a prediction of the stability trends was carried out using the kinetic and thermodynamic parameters obtained from the Arrhenius equation. The developed immunosensor as well as a commercially available enzyme-linked immunosorbent assay (ELISA) kit were successfully applied to the detection of CEA in serum samples obtained from colon cancer patients, and an excellent correlation of the levels of CEA measured was obtained. Ongoing work is looking at the incorporation of the developed biosensor into a platform for multiplexed simultaneous detection of several breast cancer related biomarkers.

Does end tidal CO2 monitoring during emergency department procedural sedation and analgesia with propofol decrease the incidence of hypoxic events? A randomized, controlled trial.

STUDY OBJECTIVE: We determine whether the use of capnography is associated with a decreased incidence of hypoxic events than standard monitoring alone during emergency department (ED) sedation with propofol. METHODS: Adults underwent ED propofol sedation with standard monitoring (pulse oximetry, cardiac and blood pressure) and capnography and were randomized into a group in which treating physicians had access to the capnography and a blinded group in which they did not. All patients received supplemental oxygen (3 L/minute) and opioids greater than 30 minutes before. Propofol was dosed at 1.0 mg/kg, followed by 0.5 mg/kg as needed. Capnographic and SpO2 data were recorded electronically every 5 seconds. Hypoxia was defined as SpO2 less than 93%; respiratory depression, as end tidal CO2 (ETCO2) greater than 50 mm Hg, ETCO2 change from baseline of 10%, or loss of the waveform. RESULTS: One hundred thirty-two subjects were evaluated and included in the final analysis. We observed hypoxia in 17 of 68 (25%) subjects with capnography and 27 of 64 (42%) with blinded capnography (P=.035; difference 17%; 95% confidence interval 1.3% to 33%). Capnography identified all cases of hypoxia before onset (sensitivity 100%; specificity 64%), with the median time from capnographic evidence of respiratory depression to hypoxia 60 seconds (range 5 to 240 seconds). CONCLUSION: In adults receiving ED propofol sedation, the addition of capnography to standard monitoring reduced hypoxia and provided advance warning for all hypoxic events.

Design and testing of a packaged microfluidic cell for the multiplexed electrochemical detection of cancer markers.

We present the rapid prototyping of electrochemical sensor arrays integrated to microfluidics towards the fabrication of integrated microsystems prototypes for point-of-care diagnostics. Rapid prototyping of microfluidics was realised by high-precision milling of polycarbonate sheets, which offers flexibility and rapid turnover of the desired designs. On the other hand, the electrochemical sensor arrays were fabricated using standard photolithographic and metal (gold and silver) deposition technology in order to realise three-electrode cells comprising gold counter and working electrodes as well as silver reference electrode. The integration of fluidic chips and electrode arrays was realised via a laser-machined double-sided adhesive gasket that allowed creating the microchannels necessary for sample and reagent delivery. We focused our attention on the reproducibility of the electrode array preparation for the multiplexed detection of tumour markers such as carcinoembryonic antigen and prostate-specific antigen as well as genetic breast cancer markers such as estrogen receptor-alpha, plasminogen activator urokinase receptor, epidermal growth factor receptor and erythroblastic leukemia viral oncogene homolog 2. We showed that by carefully controlling the electrode surface pre-treatment and derivatisation via thiolated antibodies or short DNA probes that the detection of several key health parameters on a single chip was achievable with excellent reproducibility and high sensitivity.

Comparison of octreotide and standard therapy versus standard therapy alone for the treatment of sulfonylurea-induced hypoglycemia.

STUDY OBJECTIVE: This study is designed to test the hypothesis that the administration of octreotide acetate (Sandostatin; Novartis Pharmaceuticals) in addition to standard therapy will increase serum glucose level measured at serial intervals in patients presenting to the emergency department (ED) with sulfonylurea-induced hypoglycemia compared with standard therapy alone. METHODS: This study was a prospective, double-blind, placebo-controlled trial. All adult patients who presented to the ED with hypoglycemia (serum glucose level < or = 60 mg/dL) and were found to be taking a sulfonylurea or a combination of insulin and sulfonylurea were screened for participation in the study. Study participants were randomized to receive standard treatment (1 ampule of 50% dextrose intravenously and carbohydrates orally) and placebo (1 mL of 0.9% normal saline solution subcutaneously) or standard treatment plus 1 dose of octreotide 75 microg subcutaneously. Subsequent treatment interventions were at the discretion of the inpatient internal medicine service. RESULTS: A total of 40 patients (18 placebo; 22 octreotide) were enrolled. The mean serum glucose measurement at presentation was placebo 35 mg/dL and octreotide 39 mg/dL. The mean glucose values for octreotide patients compared with placebo were consistently higher during the first 8 hours but showed no difference in subsequent hours. Mean glucose differences approached statistical significance from 1 to 3 hours and were significant from 4 to 8 hours after octreotide or placebo administration. CONCLUSION: The addition of octreotide to standard therapy in hypoglycemic patients receiving treatment with a sulfonylurea increased serum glucose values for the first 8 hours after administration in our patients. Recurrent hypoglycemic episodes occurred less frequently in patients who received octreotide compared with those who received placebo.

Integrated microfluidic platform for the electrochemical detection of breast cancer markers in patient serum samples.

A microsystem integrating electrochemical detection for the simultaneous detection of protein markers of breast cancer is reported. The microfluidic platform was realized by high precision milling of polycarbonate sheets and features two well distinguishable sections: a detection zone incorporating the electrode arrays and the fluid storage part. The detection area is divided into separate microfluidic chambers addressing selected electrodes for the measurement of samples and calibrators. The fluidic storage part of the platform consists of five reservoirs to store the reagents and sample, which are interfaced by septa. These reservoirs have the appropriate volume to run a single assay per cartridge and are manually filled. The liquids from the reservoirs are actuated by applying a positive air pressure (i.e.via a programmable syringe pump) through the septa and are driven to the detection zone via two turning valves. The application of the realised platform in the individual and simultaneous electrochemical detection of proteic cancer markers with very low detection limits are demonstrated. The microsystem has also been validated using real patient serum samples and excellent correlation with ELISA results obtained.

Electron permeable self-assembled monolayers of dithiolated aromatic scaffolds on gold for biosensor applications.

Self-assembled monolayers (SAMs) of thiolated compounds are formed by the spontaneous chemisorption of thiolate groups on metal surfaces. In biosensors, they are most commonly used to covalently immobilize a biorecognition molecule onto the surface of the transducer, thus offering the possibility of controlling the orientation, distribution, and spacing of the sensing element while reducing nonspecific interactions. In this paper, self-assembled monolayers of dithiolated derivatives of 3,5-dihydroxybenzyl alcohol containing carboxyl and hydroxyl end groups have been prepared on gold surfaces and characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Impedance measurements revealed that SAM formation is essentially completed after 3-5 h of exposure by observing the successive blocking of the faradic response of ferricyanide anion due to the adsorption of the dithiol molecules. The surface coverage of these molecules, estimated by reductive desorption experiments, was in the range of (1.1-2.8) x 10-10 mol/cm2. To demonstrate the potential of the dithiol SAM, a model system for detection of a tumor marker, prostate-specific antigen (PSA), was developed. The carboxyl groups of the SAM were succinimide-activated, and an anti-PSA antibody was covalently immobilized via amide bonds. The modified SAM was used for the label-free detection of prostate-specific antigen using EIS with a detection limit of 9 ng/mL. The results described here demonstrate that this kind of dithiol-modified SAM can be used as supports in electrochemical biosensors and the results are explained in terms of the structural features of these dithiols.

Induction of dystrophin expression by exon skipping in mdx mice following intramuscular injection of antisense oligonucleotides complexed with PEG-PEI copolymers.

Antisense oligonucleotides (AOs) with 2-O-methyl modifications can circumvent dystrophin mutations via exon skipping and, it is hoped, can become drugs for treatment of Duchenne muscular dystrophy (DMD). However, AO-based approaches are hindered by a lack of effective carriers to facilitate delivery of AOs to myonuclei. We examined whether copolymers composed of cationic poly(ethylene imine) (PEI) and polyethylene glycol (PEG) can enhance AO transfection in skeletal muscle of mdx mice. Single intramuscular injections of AO complexed with low Mw PEI2000(PEG550) copolymers into TA muscles of mdx mice resulted in widespread distribution of dystrophin-positive fibers at 3 weeks after injection, with no apparent cytotoxicity. Overall, injections of these low Mw polyplexes, which formed 250-nm aggregate particles, resulted in about sixfold more dystrophin-positive fibers than AO alone. Western analysis confirmed the dystrophin expression in these muscles. Surprisingly, injections of AO complexed with high Mw PEI25000(PEG5000) copolymers, which formed smaller nonaggregated particles, produced about threefold fewer dystrophin-positive fibers than injections of the low Mw polyplexes. We conclude that low Mw PEI2000(PEG550) copolymers function as high-capacity, nontoxic AO carriers suitable for in vivo transfection of skeletal muscle and are promising compounds for potential use in molecular therapy of DMD.