Electrochemical and scanning probe microscopic characterizations of spontaneously adsorbed organothiolate monolayers at gold

A host of significant processes in the life and physical sciences (e.g., biological recognition, heterogeneous catalysis, corrosion, and mechanical lubrication) are controlled by interfacial architectures. The origins of many of these macroscopic interfacial processes are controlled by the molecular level interactions at condensed phase interfaces. The development of methods that can detect differences in the composition and orientational disposition of these architectures at high spatial resolution is therefore technologically important. With the invention and the development of a range of techniques commonly called scanning probe microscopy (SPM), probing details of the interfaces at a molecular length scale has now become a reality. SPM is extensively used in the field of organothiolate monolayers. The relatively convenient manipulation of surface architectures and control of the interfacial properties of the monolayers have allowed many variants of SPM to map the chemical distribution of surface functional groups, exploiting a range of interfacial properties (e.g., friction and adhesion) as contrast mechanisms at nanometer length scale. In an attempt to develop a better understanding of the monolayer formation process, the spatial distribution of differing chemical functional groups within the monolayer, and the orientation of the end-groups, this dissertation described the insights gained from SPM with those from macroscopic characterization techniques such as infrared spectroscopy, contact angle measurements, and electrochemistry. Four studies are presented. Common to the body of this work is the use of voltammetric reductive desorption and variants of SPM to gain insight into the nature of the monolayer formation process as well as the resulting interface

Evaluation and validation of a PrintrLab-based LAMP assay to identify Trypanosoma cruzi in newborns in Bolivia: A proof-of-concept study

Vertical transmission of Trypanosoma cruzi represents approximately 20% of new Chagas disease cases. Early detection and treatment for women of childbearing age and newborns is a public health priority, but the lack of a simple and reliable diagnostic test remains a major barrier. We aimed to evaluate the performance of a point-of-care loop-mediated isothermal amplification (LAMP) assay for the detection of T cruzi. In this proof-of-concept study, we coupled a low-cost 3D printer repurposed for sample preparation and amplification (PrintrLab) to the Eiken T cruzi-LAMP prototype to detect vertically transmitted T cruzi, which we compared with standardised PCR and with the gold-standard algorithm (microscopy at birth and 2 months and serological study several months later). We screened pregnant women from two hospitals in the Bolivian Gran Chaco province, and those who were seropositive for T cruzi were offered the opportunity for their newborns to be enrolled in the study. Newborns were tested by microscopy, LAMP, and PCR at birth and 2 months, and by serology at 8 months.Between April 23 and Nov 17, 2018, 986 mothers were screened, among whom 276 were seropositive for T cruzi (28·0% prevalence, 95% CI 25·6-31·2). In total, 224 infants born to 221 seropositive mothers completed 8 months of follow-up. Congenital transmission was detected in nine of the 224 newborns (4·0% prevalence, 1·9-7·5) by direct microscopy observation, and 14 more cases were diagnosed serologically (6·3%, 3·6-10·3), accounting for an overall vertical transmission rate of 10·3% (6·6-15·0; 23 of 224). All microscopy-positive newborns were positive by PrintrLab-LAMP and by PCR, while these techniques respectively detected four and five extra positive cases among the remaining 215 microscopy-negative newborns. The PrintrLab-LAMP yielded a higher sensitivity than microscopy-based analysis. Considering the simpler use and expected lower cost of LAMP compared with PCR, our findings encourage its evaluation in a larger study over a wider geographical area.Fil: Rojas Panozo, Lizeth. Fundación Ciencia y Estudios Aplicados para el Desarrollo en Salud y Medio Ambiente ; BoliviaFil: Rivera Nina, Silvia. Fundación Ciencia y Estudios Aplicados para el Desarrollo en Salud y Medio Ambiente ; BoliviaFil: Wehrendt, Diana Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Casellas, Aina. Universidad de Barcelona; EspañaFil: Pinto, Lilian. Fundación Ciencia y Estudios Aplicados para el Desarrollo en Salud y Medio Ambiente ; BoliviaFil: Mendez, Susana. Universidad de Barcelona; EspañaFil: Kuo, Chi Wei. Ai Biosciences, Inc.; Estados UnidosFil: Lozano, Daniel F.. Fundación Ciencia y Estudios Aplicados para el Desarrollo en Salud y Medio Ambiente ; BoliviaFil: Ortiz, Lourdes. Universidad Autónoma Juan Misael Saracho.; BoliviaFil: Pinazo, Maria Jesus. Drugs for Neglected Diseases Initiative; BrasilFil: Picado, Albert. Foundation For Innovative New Diagnostics; SuizaFil: Sanz, Sergi. Universidad de Barcelona; EspañaFil: Abril, Marcelo. Fundación Mundo Sano; ArgentinaFil: Gascon, Joaquim. Universidad de Barcelona; EspañaFil: Wong, Season. Ai Biosciences, Inc.; Estados UnidosFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Torrico, Faustino. Fundación Ciencia y Estudios Aplicados para el Desarrollo en Salud y Medio Ambiente ; BoliviaFil: Alonso Padilla, Julio. Universidad de Barcelona; Españ

Electrochemical and scanning probe microscopic characterization of spontaneously adsorbed organothiolate monolayers at gold

This dissertation presented several results which add to the general knowledge base regarding organothiolates monolayer spontaneously adsorbed at gold films. Common to the body of this work is the use of voltammetric reductive resorption and variants of scanning probe microscopy to gain insight into the nature of the monolayer formation process as well as the resulting interface. The most significant result from this work is the success of using friction force microscopy to discriminate the end group orientation of monolayer chemisorbed at smooth gold surfaces with micrometer resolution (Chapter 4). The ability to detect the differences in the orientational disposition is demonstrated by the use PDMS polymer stamp to microcontact print an adlayer of n-alkanethiolate of length n in a predefine pattern onto a gold surface, followed by the solution deposition of a n-alkanethiol of n {+-} 1 to fill in the areas on the gold surface intentionally not coated by the stamping process. These two-component monolayers can be discriminated by using friction force microscopy which detects differences in friction contributed by the differences in the orientation of the terminal groups at surfaces. This success has recently led to the detection of the orientation differences at nanometer scale. Although the substrates examined in this work consisted entirely of smooth gold films, the same test can be performed on other smooth substrates and monolayer materials

A Rapid and Low-Cost PCR Thermal Cycler for Low Resource Settings.

Many modern molecular diagnostic assays targeting nucleic acids are typically confined to developed countries or to the national reference laboratories of developing-world countries. The ability to make technologies for the rapid diagnosis of infectious diseases broadly available in a portable, low-cost format would mark a revolutionary step forward in global health. Many molecular assays are also developed based on polymerase chain reactions (PCR), which require thermal cyclers that are relatively heavy (>20 pounds) and need continuous electrical power. The temperature ramping speed of most economical thermal cyclers are relatively slow (2 to 3 °C/s) so a polymerase chain reaction can take 1 to 2 hours. Most of all, these thermal cyclers are still too expensive ($2k to$4k) for low-resource setting uses.In this article, we demonstrate the development of a low-cost and rapid water bath based thermal cycler that does not require active temperature control or continuous power supply during PCR. This unit costs $130 to build using commercial off-the-shelf items. The use of two or three vacuum-insulated stainless-steel Thermos food jars containing heated water (for denaturation and annealing/extension steps) and a layer of oil on top of the water allow for significantly stabilized temperatures for PCR to take place. Using an Arduino-based microcontroller, we automate the "archaic" method of hand-transferring PCR tubes between water baths.We demonstrate that this innovative unit can deliver high speed PCR (17 s per PCR cycle) with the potential to go beyond the 1,522 bp long amplicons tested in this study and can amplify from templates down to at least 20 copies per reaction. The unit also accepts regular PCR tubes and glass capillary tubes. The PCR efficiency of our thermal cycler is not different from other commercial thermal cyclers. When combined with a rapid nucleic acid detection approach, the thermos thermal cycler (TTC) can enable on-site molecular diagnostics in low-resource settings

Ramping speed of the reagents measured with a thermocouple placed inside of a PCR vessel filled with water (between 60 and 95°C).

<p>Measured inside 0.2-mL tubes or glass capillary tubes filled with 20 μL or 17 μL of water, respectively.</p><p>Ramping speed of the reagents measured with a thermocouple placed inside of a PCR vessel filled with water (between 60 and 95°C).</p

Speed and sensitivity of PCR reactions demonstrated by TTC.

<p>PCR reactions to amplify 281 bp of the <i>nuc</i> gene from 2000, 200, 20, or 2 copies of <i>S</i>. <i>aureus</i> genomic DNA. <b>(A)</b> Lanes 1 to 4: PCR products from commercial thermal cycler. Lane 5: ladder. Lanes 6 to 9: PCR products from TTC with thin-walled plastic tubes, using a protocol of 2 min hot-start, followed by 40 cycles of 11 s and 17 s denaturation and annealing/extension. The 40-cycle reactions were completed in 22 min. <b>(B)</b> Lanes 1 to 4: PCR products from commercial thermal cycler. Lane 5: ladder. Lanes 6 to 9: PCR products from TTC with glass capillary tubes, using a protocol of 2 min hot-start, followed by 40 cycles of 7 s and 8 s denaturation and annealing/extension. The reactions were completed in 13 min and 3 s.</p

Major items needed to setup the TTC to perform rapid PCR amplifications.

<p>Major items needed to setup the TTC to perform rapid PCR amplifications.</p

Multiplexed PCR reactions performed with TTC.

<p>Lane 1: Ladder: 50/100/150/200/300/500/800/1500 bp. Lane 2: amplicons produced by the commercial thermal cycler (30 cycles in 75 min). Lane 3: amplicons produced by the TTC (40 cycles in 28 min).</p

PCR amplification of <i>Neisseria gonorrhoeae porA</i> pseudogene from clinical samples.

<p>Lane 1: ladder 50/100/150/200/300/500/800/1500 bp. Lanes 2 to 4: NTC and two positive samples performed with commercial cycler. Lane 5 to 7: NTC and two positive samples performed with TTC.</p

Effect of using oil as an insulation to keep water above 90°C for denaturation step during PCR.

<p>The water temperature inside the thermos drops significantly faster if oil is not used to insulate the water. Oil can maintain the denaturation and annealing/extension baths’ water temperature very well for over 1 hr. Because our typical 40-cycle reactions does not last longer than 30 min; PCR efficiency carried out by the TTC does not drop significantly even near the end of reaction when exponential growth is having its most effect.</p