Electrogenerated Chemiluminescence Biosensor Based on Functionalized Two-Dimensional Metal–Organic Frameworks for Bacterial Detection and Antimicrobial Susceptibility Assays

The emergence of antibiotic resistance has prompted the development of rapid antimicrobial susceptibility testing (AST) technologies to guide antibiotic prescription. A novel electrochemiluminescence (ECL) biosensor developed can quantitatively measure the binding between the lectin and lipopolysaccharide (LPS) on Gram-negative bacteria for bacterial determination and to characterize the antimicrobial activities of β-lactam and non-β-lactam antibiotics to normal and antibiotic-resistant bacteria. The biosensor utilizes ruthenium complex tagged concanavalin A (Ru-Con A) coated on NH2-MIL-53­(Al) interface for LPS binding measurements. The decreased ECL signal obtained was directly proportional to increasing Escherichia coli (E. coli) BL21 concentrations. The sensitivity displayed logarithmic dependence in the range of (50–5.0) × 104 cells/mL, with a detection limit of 16 cells/mL. The minimum inhibitory concentration (MIC) values of antibiotics for normal E. coli BL21 were 0.02–0.2, 2–4, 0.002–0.02, and 0.2–1 mg/L for levofloxacin hydrochloride (LVX), tetracycline (TCY), imipenem (IPM), and cefpirome (CPO), respectively. The increased MIC values (8–16 and 4 mg/L for IMP and CPO, respectively) in New Delhi metallo-β-lactamase-1 expressing E. coli BL21 (NDM-1-E. coli BL21) indicated greater resistance to β-lactams in NDM-1-E. coli BL21 compared with normal E. coli BL21. Therefore, the changed ECL signal because of binding between LPS with the lectin has a relation with the type of antibiotic and bacterial strains, making the ECL biosensor promote clinical practicability and facilitate antibiotic stewardship

Antimicrobial Susceptibility Assays Based on the Quantification of Bacterial Lipopolysaccharides via a Label Free Lectin Biosensor

A label free lectin biosensor developed in our laboratory that can quantitatively measure the binding between the lectin immobilized at the carbohydrate sensor surface and the lipopolysaccharide (LPS) on Gram-negative bacteria was demonstrated for an antibiotic susceptibility assay. The biosensor utilizes a polythiophene interface containing fused quinone moieties glycosylated to form a carbohydrate platform for the immobilization of Concanavalin A (Con A) and is capable of LPS binding measurements via orthogonal quartz crystal microbalance and electrochemical readouts (EQCM). Such orthogonal transduction provides cross-validation, better sensor sensitivity, and a large dynamic range of the measurements. We have applied this label free lectin biosensor for a new antibiotic susceptibility assay by characterizing the antimicrobial activities of various antibiotics (i.e., ciprofloxacin, ceftriaxone, and tetracycline) against Escherichia coli W1485 as a model system. The label free biosensor allows both end point and real time measurements of antibiotic effects on the bacterial cell surface LPS, which is shown to correlate to their antibiotic effects. At the end point, after 18 h incubation of bacterial cells with these three antibiotics respectively, the bacterial LPS binding signal was reduced to 23%, 27%, and 38%, respectively, for the three antibiotics, indicating that ciprofloxacin is the most effective against this E. coli strain. Real time measurements at the 1 h time point showed a similar trend with a reduction of binding to 91%, 93%, and 95%, respectively. From the binding kinetics of these measurements, the relaxation time (τ) was obtained, where higher τ value means slow binding interactions between the lectin and the bacterial LPS. The obtained order of τ, (i.e., τ_{ciprofloxacin} > τ_ceftriaxone > τ_tetracycline) again indicated that ciprofloxacin has more bactericidal activity than the other two antibiotics with the same concentrations. Thus, we are able to establish that the reduction in the binding of LPS with the lectin Con A sensor upon exposure to various antibiotics has a direct relation with the antibiotic dosages making this label free biosensor assay promising for therapeutic management of these drugs as well as for applications in antibiotic research and development

Double Covalent Coupling Method for the Fabrication of Highly Sensitive and Reusable Electrogenerated Chemiluminescence Sensors

A double covalent coupling method for the fabrication of a highly sensitive and reusable electrogenerated chemiluminescence (ECL) chemical sensor for the detection of tertiary amines and ECL aptamer-based (ECL-AB) biosensor for the detection of cocaine is reported. The ECL sensors were constructed by covalent coupling of amino-containing Ru(bpy)32+ derivatives (Ru1, Ru(bpy)32+ = tris(2,2′-bipyridyl)ruthenium(II)) or cocaine aptamer-Ru1 to the surface of a paraffin-impregnated graphite electrode that had been covalently modified with a monolayer of 4-aminobenzene sulfonic acid via electrochemical oxidations. ECL performance of the newly developed chemical sensors was evaluated using tri-n-propylamine (TPrA) and metoclopramide (MCP) as model analytes. The sensors exhibited excellent sensitivity, stability, and reproducibility with a detection limit of 30 nM for TPrA and 2.0 nM for MCP, and relative standard deviations (RSDs) of 2.1% over 90 cyclic potential cycles (0 to 1.50 V vs Ag/AgCl) and 2.6% over 45 cycles (0.60 to +1.30 V vs Ag/AgCl) at 400 mV/s for 50 nM TPrA and 200 nM MCP, respectively. For the ECL-AB biosensor, it showed an extremely low detection limit of 10 pM for cocaine, and offered a good selectivity toward cocaine, heroin, and caffeine. This detection limit was about 4−6 orders of magnitude lower than that reported on the basis of alternating current (AC) voltammetry and optical aptamer-based cocaine biosensors. Additionally, the ECL-AB biosensor was highly reusable (RSD = 2.8%, n = 7) and possessed long-term storage stability (96.8% initial ECL recovery over 21 days storage). A binding constant of 4.6 ± 0.3 × 109 M−1 between cocaine and its aptamer was estimated using an ECL based Langmuir isotherm approach. Wide ranging applications of the presently reported strategy in fabricating various chemical sensors or biosensors are expected

Glycosylation of Quinone-Fused Polythiophene for Reagentless and Label-Free Detection of <i>E. coli</i>

In this report, a new polythiophene interface is fabricated containing fused quinone moieties which are then glycosylated to form a carbohydrate platform for bacterial detection. Very importantly, this interface can be used for label-free and reagentless detection, both by electrochemical and Quartz Crystal Microbalance (QCM) transducers and by using the direct pili-mannose binding as well as Concanavalin A (Con A) mediated lipopolysaccharides (LPS)-mannose binding. The conductive polymer’s unique collective properties are very sensitive to very minor perturbations, which result in significant changes of electrical conductivity and providing amplified sensitivity and improved limits of detection (i.e., 25 cell/mL for electrochemical sensor and 50 cells/mL for QCM sensor), a widened logarithmic range of detection (i.e., 3–7 for pili-mannose binding and 2–8 for Con A mediated binding), high specificity and selectivity, and an extraordinary reliability by a mechanism of internal validation. With these analytical performances, the described biosensor is envisaged for being capable of differentiating Gram-negative bacterial strain and species, for many important applications

Figures S1 - S9 from BMX-Mediated Regulation of Multiple Tyrosine Kinases Contributes to Castration Resistance in Prostate Cancer

Figure S1. Inducible BMX knockdown suppresses CWR22RV1 tumor growth in vitro and in vivo; Figure S2. Schematic picture of BMX signaling pathway; Figure S3. The effect of BMX SiRNA smart pool on CWR22RV1 cell response to serum stimulation; Figure S4. The specificity of BMX antibody; Figure S5. Affymetrix microarray expression of BMX in PCa patients; Figure S6. BMX mRNA expression is repressed by DHT; Figure S7. BMX knockdown in CWR22RV1 cells; Figure S8. AR binding site sequence in BMX loci; Figure S9. DHT effect on expression of AR and associated proteins.</p

Supplementary Table S6 from Neoadjuvant-Intensive Androgen Deprivation Therapy Selects for Prostate Tumor Foci with Diverse Subclonal Oncogenic Alterations

Shared somatic mutations</p

Supplementary Table S5 from Neoadjuvant-Intensive Androgen Deprivation Therapy Selects for Prostate Tumor Foci with Diverse Subclonal Oncogenic Alterations

Somatic copy number alterations</p

Supplementary Table S7 from Neoadjuvant-Intensive Androgen Deprivation Therapy Selects for Prostate Tumor Foci with Diverse Subclonal Oncogenic Alterations

Unique somatic mutations</p

Supplementary Tables S1,S2,S3,S4,S9 from Neoadjuvant-Intensive Androgen Deprivation Therapy Selects for Prostate Tumor Foci with Diverse Subclonal Oncogenic Alterations

Supplementary Table S1. Clinicopathologic characteristics of microdissected cases; Supplementary Table S2. Genes up-regulated by 2-fold or more in the treated vs. untreated cases; Supplementary Table S3. Fold change in expression of AR coactivators in treated versus untreated cases; Supplementary Table S4. Genes down-regulated by 2-fold or more in the treated versus untreated cases; Supplementary Table S9. Number of genomic alterations in each focus.</p