Spotting Technology Research Articles

Smart sprayer a technology for site-specific herbicide application

Reducing the use of chemical-synthetic herbicides with site-specific application technology will play an important role in agricultural research and practice in the coming years and decades. This paper presents a sprayer for site-specific weed control in row crops using weed detection technology based on image analysis and spot application using weed detection data. Two small plot trials in sugar beet and maize in 2021 and seven On-farm field trials in sugar beet, sunflower and maize in 2022 are presented. A pair-wise comparison of broadcast application, untreated control and spotting application were realized. A zero-tolerance strategy for weeds was used as a spotting application in all experiments. Herbicide savings from 10% to 55% were realized without a significant decrease in efficacy in weed control for the spotting application compared to the broadcast application. The weed control efficacy for broadcast application varied from 70 to 100%, while for the spotting application 72–99%. Weed control between broadcast and spotting did not differ significantly within a trial, except one experiment. Application mistakes caused the partly observed weed control of under 80%. Yield tends to be higher for all spotting treatments compared to broadcast application. The presented system provides a robust spotting technology for effective weed management with lower environmental risk and a positive effect on biodiversity.

Abstract 762: Organo-on-pillar based anticancer drug screening platform for ovarian cancer

Abstract Previously preclinical models for customized drug screening and precision oncology are being developed to replace animal models. Advances in biocompatible organoid technology have made it possible to culture cancer organoids from human tumor tissues. Cancer organoids are easy to proliferate, and large-scale drug screening platforms can be developed in that they can reproducibly repeat experiments with various concentrations of drugs. In this study, we developed automated extracellular matrix integrated, three-dimensional, patient-derived cell spotting technology and pillar-based cancer organoid culture system for anticancer drug screening platform as a preclinical model which able to contribute for prediction of therapeutic responses and selection of optimal next line anticancer drugs. In case of ovarian cancer, about 70-80% of patients are firstly diagnosed, but their 5-year survival rate is still under 50%. To increase the 5-year survival rate, raising therapeutic responses is urgently needed by effective drug treatment rather than standard therapy. For drugs selected through a drug screening platform to be applied to actual clinical practice, standard test methods must be set up, and clinical results and organoid-based treatment responses must be clearly compared and verified. For validation above, we confirmed the organoid on pillar-based drug testing platform by using surgical specimens from 109 ovarian cancer patients from SMC (Samsung medical center). Primary cells with extracellular matrix spotted and cultured on 384 pillar plates by ASFA spotter (MBD Inc.). After organoid formation on pillars, anticancer drug screening was performed for 14 drug combinations using cancer organoids derived from the ascites of patients. Cell viabilities and drug responses were calculated by ATP measurement and staining of intracellular Calcein then. Particularly in this study, parallel with drug treatment, organoid growth rate was measured by comparing the average areas, processed by ASFA cell analyzer (MBD Inc.), of organoids at drug treatment and termination day. Finally anticancer drug sensitivity index was calculated by combination of the drug response of AUC and respective organoid growth rate, then it was verified by matching with the clinical responses of 1st chemotherapies after surgery. As a results, the drug susceptibility index was predicted patients of recurrence within 6-month after 1st chemotherapy by our drug screening platform with increased sensitivity and specificity. This anticancer drug screening platform raised the recurrence prediction about 90% compared to existing drug sensitivity index such as IC50 or AUC, and through this, this platform could contribute to precision medicine in ovarian cancer patients. Therefore, it can be preclinical model used to screen the optimal drug for customized treatment using patient-specific individual organoids. Citation Format: Hye Ryeong Jun, Sung Hun Ju, Jung Eun Kim, Dong Woo Lee, Bosung Ku, Joseph Noh, Jeogn-Won Lee, Hyun Ju Kang, Jin-Ku Lee. Organo-on-pillar based anticancer drug screening platform for ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 762.

Research on Deep Coalbed Methane Localized Spotting and Efficient Permeability Enhancement Technology

To solve the bottleneck problem of low deep coal seam permeability and difficult coalbed methane (CBM) mining. Combining hydraulic splitting technology and directional drilling technology, a directional hydraulic splitting enhancement method of deep CBM mining was proposed. The selection equation for the directional hydraulic splitting of deep coalbed was constructed. The numerical simulation reveals the variation in coal fractures around different split angles. The split angle under the maximum coal damage effect was obtained. It was found that the combined effect of the double crack damage disturbance region led to reciprocal stress fluctuations during crack development and, eventually, the formation of a zigzag fracture. The larger the splitting angle, the larger the fissure development length and the larger the coal-damaged area. A double crack takes 25% less time to complete propagation than a single crack. When the splitting angle is 90°, the disturbed area occupies 2/3 of the area around the borehole, and the overall fracturing effect is the best. In the application process, the new directional hydraulic splitting technology can increase CBM mining by 5.08%, greatly improve CBM mining efficiency, and reduce the coal mining risk, which is of great significance to the project.

Low-Power Audio Keyword Spotting Using Tsetlin Machines

The emergence of artificial intelligence (AI) driven keyword spotting (KWS) technologies has revolutionized human to machine interaction. Yet, the challenge of end-to-end energy efficiency, memory footprint and system complexity of current neural network (NN) powered AI-KWS pipelines has remained ever present. This paper evaluates KWS utilizing a learning automata powered machine learning algorithm called the Tsetlin Machine (TM). Through significant reduction in parameter requirements and choosing logic over arithmetic-based processing, the TM offers new opportunities for low-power KWS while maintaining high learning efficacy. In this paper, we explore a TM-based keyword spotting (KWS) pipeline to demonstrate low complexity with faster rate of convergence compared to NNs. Further, we investigate the scalability with increasing keywords and explore the potential for enabling low-power on-chip KWS.

Combinatorial programming of human neuronal progenitors using magnetically-guided stoichiometric mRNA delivery.

Identification of optimal transcription factor expression patterns to direct cellular differentiation along a desired pathway presents significant challenges. We demonstrate massively combinatorial screening of temporally-varying mRNA transcription factors to direct differentiation of neural progenitor cells using a dynamically-reconfigurable magnetically-guided spotting technology for localizing mRNA, enabling experiments on millimetre size spots. In addition, we present a time-interleaved delivery method that dramatically reduces fluctuations in the delivered transcription factor copy numbers per cell. We screened combinatorial and temporal delivery of a pool of midbrain-specific transcription factors to augment the generation of dopaminergic neurons. We show that the combinatorial delivery of LMX1A, FOXA2 and PITX3 is highly effective in generating dopaminergic neurons from midbrain progenitors. We show that LMX1A significantly increases TH-expression levels when delivered to neural progenitor cells either during proliferation or after induction of neural differentiation, while FOXA2 and PITX3 increase expression only when delivered prior to induction, demonstrating temporal dependence of factor addition.

Low-Volume Label-Free Detection of Molecule-Protein Interactions on Microarrays by Imaging Reflectometric Interferometry.

This system allows the high-throughput protein interaction analysis on microarrays. We apply the interference technology 1λ-imaging reflectometric interferometry (iRIf) as a label-free detection method and create microfluidic flow cells in microscope slide format for low reagent consumption and lab work compatibility. By now, most prominent for imaging label-free interaction analyses on microarrays are imaging surface plasmon resonance (SPR) methods, quartz crystal microbalance, or biolayer interferometry. SPR is sensitive against temperature drifts and suffers from plasmon crosstalk, and all systems lack array size (maximum 96 spots). Our detection system is robust against temperature drifts. Microarrays are analyzed with a spatial resolution of 7 µm and time resolution of ≤50 fps. System sensitivity is competitive, with random noise of <5 × 10-5 and baseline drift of <3 × 10-6. Currently available spotting technologies limit array sizes to ~4 spots/mm2 (1080 spots/array); our detection system would allow ~40 spots/mm2 (10,800 spots/array). The microfluidic flow cells consist of structured PDMS inlays sealed by versatilely coated glass slides immobilizing the microarray. The injection protocol determines reagent volumes, priming rates, and flow cell temperatures for up to 44 reagents; volumes of ≤300 µL are validated. The system is validated physically by the biotinylated bovine serum albumin streptavidin assay and biochemically by thrombin aptamer interaction analysis, resulting in a KD of ~100 nM.

High-resolution droplet-based fractionation of nano-LC separations onto microarrays for MALDI-MS analysis.

We present a robust droplet-based device, which enables the fractionation of ultralow flow rate nanoflow liquid chromatography (nano-LC) eluate streams at high frequencies and high peak resolution. This is achieved by directly interfacing the separation column to a micro T-junction, where the eluate stream is compartmentalized into picoliter droplets. This immediate compartmentalization prevents peak dispersion during eluate transport and conserves the chromatographic performance. Subsequently, nanoliter eluate fractions are collected at a rate of one fraction per second on a high-density microarray to retain the separation with high temporal resolution. Chromatographic separations of up to 45 min runtime can thus be archived on a single microarray possessing 2700 sample spots. The performance of this device is demonstrated by fractionating the separation of a tryptic digest of a known protein mixture onto the microarray chip and subsequently analyzing the sample archive using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Resulting peak widths are found to be significantly reduced compared to standard continuous flow spotting technologies as well as in comparison to a conventional nano-LC-electrospray ionization-mass spectrometry interface. Moreover, we demonstrate the advantage of our high-definition nanofractionation device by applying two different MALDI matrices to all collected fractions in an alternating fashion. Since the information that is obtained from a MALDI-MS measurement depends on the choice of MALDI matrix, we can extract complementary information from neighboring spots containing almost identical composition but different matrices.

A keyword retrieval system for historical Mongolian document images

In this paper, we propose a keyword retrieval system for locating words in historical Mongolian document images. Based on the word spotting technology, a collection of historical Mongolian document images is converted into a collection of word images by word segmentation, and a number of profile-based features are extracted to represent word images. For each word image, a fixed-length feature vector is formulated by obtaining the appropriate number of the complex coefficients of discrete Fourier transform on each profile feature. The system supports online image-to-image matching by calculating similarities between a query word image and each word image in the collection, and consequently, a ranked result is returned in descending order of the similarities. Therein, the query word image can be generated by synthesizing a sequence of glyphs when being retrieved. By experimental evaluations, the performance of the system is confirmed.

Microcontact printing and microspotting as methods for direct protein patterning on plasma deposited polyethylene oxide: application to stem cell patterning

Two methods for protein patterning on antifouling surfaces have been applied to analyze the density and bioactivity of the proteins after deposition. Microcontact printing has been used as a technique to transfer fibronectin through conformal contact, while piezoelectric deposition has been employed as a non-contact technique for producing arrays of fibronectin (FN). Plasma deposited polyethylene oxide-like (PEO-like) films have been used as non-fouling background to achieve the bioadhesive/biorepellent surface contrast. Both patterning methods allow the direct fabrication of protein arrays on a non-fouling substrate, and the subsequent formation of a pattern of stem cells by cell attachment on the arrayed substrates. Microcontact printing produced fully packed homogeneous fibronectin patterns, much denser than microspotted patterns. Both printing and spotting technologies generated functional protein arrays, their bioactivity being primarily modulated by the density of the deposited protein layer. Optimization of the FN parameters used for deposition has lead to the achievement of high-quality microarrays with large population of neural stem cells immobilized in the patterns in serum-free conditions, where cells exhibit a more homogeneous starting population and factors influencing fate decisions can be more easily tracked. The immunorecognition of fibronectin targeted antibodies, as well as the cell density, increase with the protein density up to a saturation point. Over 100 ng/cm² of fibronectin on the surface leads to a decrease in the number of attached cells and a raise of cell spreading.

New Approaches for Carbon Nanotubes-Based Biosensors and Their Application to Cell Culture Monitoring

Amperometric biosensors are complex systems and they require a combination of technologies for their development. The aim of the present work is to propose a new approach in order to develop nanostructured biosensors for the real-time detection of multiple metabolites in cell culture flasks. The fabrication of five Au working electrodes onto silicon substrate is achieved with CMOS compatible microtechnology. Each working electrode presents an area of 0.25 mm², so structuration with carbon nanotubes and specific functionalization are carried out by using spotting technology, originally developed for microarrays and DNA printing. The electrodes are characterized by cyclic voltammetry and compared with commercially available screen-printed electrodes. Measurements are carried out under flow conditions, so a simple fluidic system is developed to guarantee a continuous flow next to the electrodes. The working electrodes are functionalized with different enzymes and calibrated for the real-time detection of glucose, lactate, and glutamate. Finally, some tests are performed on surnatant conditioned medium sampled from neuroblastoma cells (NG-108 cell line) to detect glucose and lactate concentration after 72 hours of cultivation. The developed biosensor for real-time and online detection of multiple metabolites shows very promising results towards circuits and systems for cell culture monitoring.

Deubiquitylase, DeSUMOylase, and DeISGylase Activity Microarrays for Assay of Substrate Preference and Functional Modifiers

Microarray-based proteomics expanded the information potential of DNA arrays to the level of protein translation and interaction, but so far, not much beyond. Although enzymatic activity from immobilized proteins has been reliably studied using surface plasmon resonance, a microarray of catalytically competent enzymes would facilitate high throughput, parallel study of their function. The ability to localize activity from soluble substrates has frustrated development of such an array. Here, we report the novel use of previously developed, highly specific suicide substrates for three families of enzymes: deubiquitylases, deSUMOylases, and deISGylases. We show specificity of each family to its cognate substrate, and demonstrate utility of the array in a secondary screen of small molecule inhibitors.

Essential tactics of tissue preparation and matrix nano-spotting for successful compound imaging mass spectrometry

The ultimate goal of MALDI-Imaging Mass Spectrometry (MALDI-IMS) is to achieve spatial localization of analytes in tissue sections down to individual tissue compartments or even at the level of a few cells. With compound tissue imaging, it is possible to track the transportation of an unlabelled, inhaled reference compound within lung tissue, through the application of MALDI-IMS. The procedure for isolation and preparation of lung tissues is found to be crucial in order to preserve the anatomy and structure of the pulmonary compartments. To avoid delocalization of analytes within lung tissue compartments we have applied an in-house designed nano-spotter, based on a microdispenser mounted on an XY table, of which movement and spotting functionality were fully computer controlled. We demonstrate the usefulness of this platform in lung tissue sections isolated from rodent in vivo model, applied to compound tissue imaging as exemplified with the determination of the spatial distribution of (1alpha,2beta,4beta,7beta)-7-[(hydroxidi-2-thienylacetyl)oxy]-9,9-dimethyl-3-oxa-9-azoniatricyclo[3.3.1.0(2,4)]nonane, also known as tiotropium. We provide details on tissue preparation protocols and sample spotting technology for successful identification of drug in mouse lung tissue by using MALDI-Orbitrap instrumentation.

Living-Cell Microarrays

Living cells are remarkably complex. To unravel this complexity, living-cell assays have been developed that allow delivery of experimental stimuli and measurement of the resulting cellular responses. High-throughput adaptations of these assays, known as living-cell microarrays, which are based on microtiter plates, high-density spotting, microfabrication, and microfluidics technologies, are being developed for two general applications: (a) to screen large-scale chemical and genomic libraries and (b) to systematically investigate the local cellular microenvironment. These emerging experimental platforms offer exciting opportunities to rapidly identify genetic determinants of disease, to discover modulators of cellular function, and to probe the complex and dynamic relationships between cells and their local environment.

Biosensors: Biomolecule Spotting Technologies and Commercially Available Devices

Biosensors: Biomolecule Spotting Technologies and Commercially Available Devices

Improved continuous-flow print head for micro-array deposition

Limitations in depositing ligands using conventional micro-array pin spotting have hindered the application of surface plasmon resonance imaging (SPRi) technology. To address these challenges we introduce a modification to our continuous-flow micro-spotting technology that improves ligand deposition. Using Flexchip™ protein A/G and neutravidin capturing surfaces, we demonstrate that our new microfluidic spotter requires 1000 times less concentrated antibodies and biotinylated ligands than is required for pin spotting. By varying the deposition flow rate, we show that the design of our tip overlay flow cell is efficient at delivering sample to the substrate surface. Finally, contact time studies show that it is possible to capture antibodies and biotinylated ligands at concentrations of less than 0.1 ug/ml and 100 pM, respectively. These improvements in spotting technology will help to expand the applications of SPRi systems in areas such as antibody screening, carbohydrate arrays, and biomarker detection.

Advanced Nanotechnological Approaches for Designing Protein-Based &amp;#x201C;Lab-on-Chips&amp;#x201D; Sensors on Porous Silicon Wafer

In this article, we will review the more recent patented approaches related to the design and development of micro- and nano-patterns of biomolecules on solid substrates for the realization of innovative biochips, including inkjet and spotting technology, and Scanning Probe Methods In addition, we will report on some important patents based on the use of porous materials as substrates, exploiting the large specific surface for the design of highly sensitive biodevices. The main advantages and drawbacks related to each technological approach to the biochips fabrication will be pointed out, and future perspectives in the field will be discussed.

Printing of protein microarrays via a capillary-free fluid jetting mechanism

Current proteomics experiments rely upon printing techniques such as ink jet, pin, or quill arrayers that were developed for the creation of cDNA microarrays. These techniques often do not meet the requirements needed for successful spotting of proteins to perform high-throughput, array-based proteomic profiling. Biological laser printing (BioLP) is a spotting technology that does not rely on solid pins, quill pins, or capillary-based fluidics. The non-contact mechanism of BioLP utilizes a focused laser pulse to transfer protein solutions, thereby eliminating the potential for orifice clogging, air bubbles, and unnecessary volume loss potentially encountered in commercially available spotting technologies. The speed and spot-to-spot reproducibility of BioLP is comparable to other techniques, while the minimum spot diameter and volume per printed droplet is significantly less at 30 microm and approximately 500 fL, respectively. The transfer of fluid by BioLP occurs through a fluid jetting mechanism, as observed by high-speed images of the printing process. Arraying a solution of BSA with subsequent immunodetection demonstrates the reproducible spotting of protein in an array format with CVs of <3%. Printing of the enzyme alkaline phosphatase followed by a positive reaction with a colorimetric substrate demonstrates that functional protein can be spotted using this laser-based printer.

Direct microcontact printing of oligonucleotides for biochip applications

BackgroundA critical step in the fabrication of biochips is the controlled placement of probes molecules on solid surfaces. This is currently performed by sequential deposition of probes on a target surface with split or solid pins. In this article, we present a cost-effective procedure namely microcontact printing using stamps, for a parallel deposition of probes applicable for manufacturing biochips.ResultsContrary to a previous work, we showed that the stamps tailored with an elastomeric poly(dimethylsiloxane) material did not require any surface modification to be able to adsorb oligonucleotides or PCR products. The adsorbed DNA molecules are subsequently printed efficiently on a target surface with high sub-micron resolution. Secondly, we showed that successive stamping is characterized by an exponential decay of the amount of transferred DNA molecules to the surface up the 4th print, then followed by a second regime of transfer that was dependent on the contact time and which resulted in reduced quality of the features. Thus, while consecutive stamping was possible, this procedure turned out to be less reproducible and more time consuming than simply re-inking the stamps between each print. Thirdly, we showed that the hybridization signals on arrays made by microcontact printing were 5 to 10-times higher than those made by conventional spotting methods. Finally, we demonstrated the validity of this microcontact printing method in manufacturing oligonucleotides arrays for mutations recognition in a yeast gene.ConclusionThe microcontact printing can be considered as a new potential technology platform to pattern DNA microarrays that may have significant advantages over the conventional spotting technologies as it is easy to implement, it uses low cost material to make the stamp, and the arrays made by this technology are 10-times more sensitive in term of hybridization signals than those manufactured by conventional spotting technology.

Subnanoliter enzymatic assays on microarrays

Many areas of research today are based on enzymatic assays most of which are still performed as enzyme-linked immunosorbent assays in microtiter plates. The demand for highly parallel screening of thousands of samples eventually led to a miniaturization and automation of these assays. However, the final transfer of enzymatic assays from a microtiter-based technology to microarrays has proven to be difficult for various reasons, such as the inability to maintain unbound reaction products on the spot of reaction or the missing capability of multiplexing. Here, we have conducted multiplex enzymatic assays in subnanoliter volumes on a single microarray using the multiple spotting technology. We were able to measure enzymatic activity with a sensitivity down to 35 enzyme molecules, applying only conventional flat microarray surfaces and standard microarray hardware. We have performed assays of inhibition and applied this format for the detection of prognostic markers, such as cathepsin D. The new approach allows the rapid and multiplex screening of thousands of samples on a single microarray with applications in drug screening, metagenomics, and high-throughput enzyme assays.

“The Spotting Accelerator™”, Customizable Head Assembly for Advanced Microarraying

Microarraying technologies have emerged as key tools for determining genomic expression. The quality of gene arrays is reliant on spotting pins for production of uniform spots, which are consistent in volume, shape, size and alignment. Point Technologies, Inc., has developed an advanced spotting technology. Emphasis has been placed on producing the “perfect spots” customized to each microarraying application. Uniform surface texturing such as highly polished or matte finishes, and hydrophilic/hydrophobic zonal coating is produced using Point Technologies' proprietary micro-machining technology. Micro-fabrication also permits the production of advanced pin designs, and high density, high throughput print-head assemblies of 1536 pins and greater. Point Technologies specializes in the micro-electrochemical machining of spotting pins in any size, shape and configuration. Presented data will demonstrate spotting pin design solutions, such as: variety of tip dimensions, surface finishes, specialized coatings, and material options instrumental in producing the ideal spot size, shape and spot density during extended usage.