Tissue Multiplex Analyte Detection in Anatomic Pathology - Pathways to Clinical Implementation.

Multiplex tissue analysis has revolutionized our understanding of the tumor microenvironment (TME) with implications for biomarker development and diagnostic testing. Multiplex labeling is used for specific clinical situations, but there remain barriers to expanded use in anatomic pathology practice. We review immunohistochemistry (IHC) and related assays used to localize molecules in tissues, with reference to United States regulatory and practice landscapes.

The InSituPlex Staining Method for Multiplexed Immunofluorescence Cell Phenotyping and Spatial Profiling of Tumor FFPE Samples.

Multiplexed immunohistochemistry (mIHC) enables the detection, quantification, and localization of many markers within cell or tissue samples, leading to a better understanding of the architecture of a disease at the cellular level. Current mIHC techniques involve long staining and assay times, require dedicated and/or captive instrumentation, and entail tedious assay optimization, hindering their establishment as routine methods. Here, we demonstrate the use of the InSituPlex method for spatial profiling of immuno-oncology targets in FFPE tumor tissue with the UltiMapper™ I/O PD-L1 multiplex assay.

Protein Counting in Single Cancer Cells.

The cell is the basic unit of biology and protein expression drives cellular function. Tracking protein expression in single cells enables the study of cellular pathways and behavior but requires methodologies sensitive enough to detect low numbers of protein molecules with a wide dynamic range to distinguish unique cells and quantify population distributions. This study presents an ultrasensitive and automated approach for quantifying phenotypic responses with single cell resolution using single molecule array (SiMoA) technology.

Measuring atomic emission from beacons for long-distance chemical signaling.

In an effort to exploit chemistry for information science, we have constructed a system to send a message powered by a combustion reaction. Our system uses the thermal excitation of alkali metals to transmit an encoded signal over long distances. A message is transmitted by burning a methanol-soaked cotton string embedded with combinations of high, low, or zero levels of potassium, rubidium, and/or cesium ions.

Assessing the stochastic intermittency of single quantum dot luminescence for robust quantification of biomolecules.

Single molecule detection schemes promise that one has the ability to reach the ultimate limit of detection: one molecule. In this paper, we use the stochastic luminescence of single semiconductor nanocrystals (quantum dots, QDs) to detect and localize particles as digital counts. These digital counts can be correlated to the concentration of analytes in solution.

Dynamic microbead arrays for biosensing applications.

In this paper we present the development of an optical tweezers platform capable of creating on-demand dynamic microbead arrays for the multiplexed detection of biomolecules. We demonstrate the use of time-shared optical tweezers to dynamically assemble arrays of sensing microspheres, while simultaneously recording fluorescence signals in real time. The detection system is able to achieve multiplexing by using quantum dot nanocrystals as both signaling probes and encoding labels on the surface of the trapped microbeads.

InfoBiology by printed arrays of microorganism colonies for timed and on-demand release of messages.

This paper presents a proof-of-principle method, called InfoBiology, to write and encode data using arrays of genetically engineered strains of Escherichia coli with fluorescent proteins (FPs) as phenotypic markers. In InfoBiology, we encode, send, and release information using living organisms as carriers of data. Genetically engineered systems offer exquisite control of both genotype and phenotype.

Preparation and characterization of silver substrates coated with antimony-doped SnO2 thin films for surface plasmon resonance studies.

This paper reports on the preparation of silver/antimony-doped tin oxide (Ag/SnO(2):Sb) hybrid interfaces using magnetron sputtering and their characterization. The influence of the Sn target composition (doping with 2 or 5% Sb) on the electrochemical and electrical characteristics of the hybrid interface was investigated using X-ray photoelectron spectroscopy (XPS), sheet resistance measurements, cyclic voltammetry, scanning tunneling microscopy (STM) and surface plasmon resonance (SPR). The best interface in terms of electrical conductivity and SPR signal is a hybrid interface with a 8.

Selective detection of hexachromium ions by localized surface plasmon resonance measurements using gold nanoparticles/chitosan composite interfaces.

Selective removal of hexavalent chromium ions from aqueous solutions using a chitosan/gold nanoparticles composite film was demonstrated. Localized surface plasmon resonance (LSPR) was used to measure the interface stability and detect the incorporation of chromium ions over time. The effects of pH, ethylenediaminetetraacetic acid (EDTA), and various foreign ions such as trivalent chromium, sodium, calcium, phosphate, sulfate and chloride on the adsorption of hexavalent chromium were investigated.

Electrochemical impedance spectroscopy and surface plasmon resonance studies of DNA hybridization on gold/SiOx interfaces.

The use of Au/SiO(x) interfaces for the investigation of DNA hybridization using electrochemical impedance spectroscopy (EIS) and surface plasmon resonance (SPR) simultaneously is demonstrated. Standard glass chemistry was used to link single-stranded DNA (ss-DNA) on aldehyde-terminated Au/SiO(x) interfaces. The layer thickness and amount of grafted oligonucleotides (ODNs) were calculated from SPR on the basis of a multilayer system of glass/Ti/Au/SiO(x)/grafted molecule.

Gold island films on indium tin oxide for localized surface plasmon sensing.

Mechanically, chemically and optically stable gold island films were prepared on indium tin oxide (ITO) substrates by direct thermal evaporation of thin gold films (2-6 nm) without the need for pre- or post-coating. The effect of mild thermal annealing (150 °C, 12 h) or short high temperature annealing (500 °C, 1 min) on the morphology of the gold nanostructures was investigated. ITO covered with 2 nm gold nanoislands and annealed at 500 °C for 1 min was investigated for its ability to detect the adsorption of biotinylated bovine serum albumin using local surface plasmon resonance (LSPR), and its subsequent molecular recognition of avidin.

Localized electropolymerization on oxidized boron-doped diamond electrodes modified with pyrrolyl units.

This paper describes the functionalization of oxidized boron-doped diamond (BDD) electrodes with N-(3-trimethoxysilylpropyl)pyrrole (TMPP) and the influence of this layer on the electrochemical transfer kinetics as well as on the possibility of forming strongly adhesive polypyrrole films on the BDD interface through electropolymerization. Furthermore, localized polymer formation was achieved on the TMPP-modified BDD interface using the direct mode of a scanning electrochemical microscope (SECM) as well as an electrochemical scanning near-field optical microscope (E-SNOM). Depending on the method used polypyrrole dots with diameters in the range of 1-250 microm are electrogenerated.