Ultrasensitive evanescent wave optical fiber aptasensor for online, continuous, type-specific detection of sulfonamides in environmental water.

Sensors capable for online continuous monitoring of total sulfonamides in environmental waters are highly desired due to their adverse effects on ecosystem, unexpected concentration fluctuation, and diversity. At present, no sensor with this capability has been reported. In this study, we evaluated the cross reactivity (CR) of the previously reported sulfadimethoxine-binding aptamer using DNase I assay and found that the aptamer was type-specific to sulfonamides. We then fabricated the first type-specific sulfonamide sensor, where the aptamer was immobilized on the optical fiber of the evanescent wave sensor, followed by the surface coating with Tween 80. The competitive binding of sulfonamides and Cy5.5 labeled complementary DNA enabled the low femtomolar to picomolar sensitivity and the detection of total 14 sulfonamides spiked in the lake water. The sensor also exhibited high selectivity, regeneration capability (40 cycles), stability (65 days), and short detection time (5 min). In addition, we found that the CRs were greatly dependent on the buffer composition. By performing the parallel detections in two buffers, the sensors detected 18 out of the 24 sulfonamides with the diversity coverage higher than commercial ELISA kits. Our aptasensor fills the technical gap for continuous monitoring of total sulfonamides in environmental waters.

Strong additive and synergistic effects of polyoxyethylene nonionic surfactant-assisted protein MALDI imaging mass spectrometry.

Protein MALDI imaging mass spectrometry (MALDI-IMS) holds a great promise to acquire spatial distribution information of proteins on biological tissue, but it suffers from the small number of proteins detected by direct MALDI-IMS detection. Ionic surfactants have been extensively used for protein extraction to improve the number of proteins detected in tissue samples by LC-MS analysis, but seldom by direct MALDI-IMS detection. Nonionic surfactants are milder than ionic surfactants and protein native structures are remained after extraction, which favors the spatial resolution of direct MALDI-IMS. However, nonionic surfactants are less effective than ionic surfactants. In this report, we utilized polyoxyethylene nonionic surfactants (PNS) to pre-incubate the tissue section, followed by the on-tissue trypsin digestion and then direct MALDI detection of in-situ formed peptides. For the first time, we observed that the additive effect of PNS and the synergistic effect of the mixed PNS in improving the number of peptides detected. Specifically, the peptides detected were 73.0-90.7% distinct when the different PNS (Tween 80 or Triton X-100 alone or their mixture) was used. Taking advantage of this additive effect, the 96 proteins including 12 transmembrane proteins were detected, corresponding to a ~10-fold improvement compared to MALDI-IMS without surfactant. When the mixed surfactants were used to replace Tween 80 and Triton X-100 alone, the optimized surfactant concentration decreased 20-100-fold and the number of peptides detected with m/z > 2500 Da was improved 15-fold. The additive and synergistic effects of PNS suggested that the interaction mode between each PNS and proteins is highly variable. Benefiting from the strong additive effect and diversity of PNS, further improvement of the number of proteins detected by MALDI-IMS is clearly feasible.

Amplification-by-Polymerization in Biosensing for Human Genomic DNA Detection.

A polymerization reaction was employed as a signal amplification method to realize direct visualization of gender-specific DNA extracted from human blood in a polymerase chain reaction (PCR)-free fashion. Clear distinction between X and Y chromosomes was observed by naked eyes for detector-free sensing purposes. The grown polymer films atop X and Y chromosomes were quantitatively measured by ellipsometry for thickness readings. Detection assays have been optimized for genomic DNA recognition to a maximum extent by varying the selection of the proper blocking reagents, the annealing temperature, and the annealing time. Traditional PCR and gel electrophoresis for amplicon identification were conducted in parallel for performance comparison. In the blind test for blood samples examined by the new approach, 25 out of 26 were correct and one was false negative, which was comparable to, if not better than, the PCR results. This is the first time our amplification-by-polymerization technique is being used for chromosome DNA analysis. The potential of adopting the described sensing technique without PCR was demonstrated, which could further promote the development of a portable, PCR-free DNA sensing device for point-of-need applications.

Zeta-potential data reliability of gold nanoparticle biomolecular conjugates and its application in sensitive quantification of surface absorbed protein.

Zeta potentials (ZP) of gold nanoparticle bioconjugates (AuNP-bios) provide important information on surface charge that is critical for many applications including drug delivery, biosensing, and cell imaging. The ZP measurements (ZPMs) are conducted under an alternative electrical field at a high frequency under laser irradiation, which may strongly affect the status of surface coating of AuNP-bios and generate unreliable data. In this study, we systemically evaluated the ZP data reliability (ZPDR) of citrate-, thiolated single stranded DNA-, and protein-coated AuNPs mainly according to the consistence of ZPs in the repeated ZPMs and the changes of the hydrodynamic size before and after the ZPMs. We found that the ZPDR was highly dependent on both buffer conditions and surface modifications. Overall, the higher ionic strength of the buffer and the lower affinity of surface bounders were related with the worse ZPDR. The ZPDR of citrate-coated AuNP was good in water, but bad in 10mM phosphate buffer (PB), showing substantially decrease of the absolute ZP values after each measurement, probably due to the electrical field facilitated adsorption of negatively charged phosphate ions on AuNPs. The significant desorption of DNAs from AuNP was observed in the PB containing medium concentration of NaCl, but not in PB. The excellent ZPDR of bovine serum albumin (BSA)-coated AuNP was observed at high salt concentrations and low surface coverage, enabling ZPM as an ultra-sensitive tool for protein quantification on the surface of AuNPs with a single molecule resolution.

Core-shell Au nanoparticle formation with DNA-polymer hybrid coatings using aqueous ATRP.

We report here a direct surface-grafting approach to forming DNA-containing polymer shells outside of Au nanoparticles using aqueous atom transfer radical polymerization (ATRP). In this approach, DNA molecules were immobilized on Au particles to introduce ATRP initiators on the surface. The same DNA molecules also acted as particle stabilizers through electrostatic repulsion and allowed particles to stay suspended in water. The immobilized ATRP initiators prompted polymer chain growth under certain conditions to form thick polymer shells outside of the particles. The formation of DNA-polymer hybrids outside of Au nanoparticles was characterized using absorption spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM), and gel electrophoresis. The presence of thick polymer shells improved particle stability in high ionic strength media, whereas particles with the DNA coating only aggregated. A visible color difference between these two particle solutions was clearly observed, providing the basis for DNA sensing in homogeneous solutions.

Radical polymerization in biosensing.

This review briefly summarizes recently published work on radical polymerization in biosensor-related applications. Advancements in surface modification aimed at improving sensor biocompatibility and reducing nonspecific background noises are discussed. Direct applications of polymers as one of the key sensing elements in which they are used either as detection probes for the biomolecular binding events or as signal transducers to amplify sensing signals are detailed. Initial applications of radical polymerization reactions in biosensing are evident and appear promising.

Detection of DNA point mutation by atom transfer radical polymerization.

We report here a new DNA detection method in which polymer growth in atom transfer radical polymerization (ATRP) is used as a means to amplify detection signals. In this method, DNA hybridization and ligation reactions led to the attachment of ATRP initiators on a solid surface where specific DNA sequences were located. These initiators subsequently triggered the growth of poly(hydroxyethyl methacrylate) (PHEMA) at the end of immobilized DNA molecules and formed polymer brushes. The formation of PHEMA altered substrate opacity, rendering the corresponding spots readily distinguishable to the naked eye. A second ATRP reaction to form branched polymers on the surface drastically improved the visibility of DNA hybridization and significantly shortened the detection time. The resulting polymer film was characterized using infrared spectroscopy, ellipsometry, contact angle measurements, and atomic force microscopy. Direct visualization of 1 fmol of target DNA molecules of interest was demonstrated. A proof-of-principle experiment to detect DNA point mutation was conducted. The perfectly matched DNA targets were distinctively differentiated from those with mutations. The demonstrated capability to detect DNA mutation with direct visualization laid the groundwork for the future development of detector-free testing kits in single-nucleotide polymorphism screenings.