Characterization of the flow dynamics in Dx-FS. a) Flow rate versus angular rotation frequency under FAST and conventional conditions. Bacterial cell enrichment in Dx-FS. a) 1 ml of urine (left panel) with bacterial cells (green) is processed using Dx-FS. The color intensity was measured immediately after the bacterial cell detection test in Dx-FS (45 minutes) or after 24 hours (24 hours) at room temperature and no significant difference was observed (N = 3).
Schematic of the simple flow through array membrane integrated Dx-FS and concept of RPA-based detection.
Introduction
Diagnostics
- Evolving diagnostic needs
- Point of care test
The outcomes by increasing are measured with life expectancy, its identified instruments UN Millennium Development Goals (MDGs) have helped to promote the development of new POCT technologies in the last 10-15 years; these new technologies have done well in improving life expectancies for people from different parts of the world. Increase in migration and evaluation of immune systems has caused epidemics in various parts of the world, this has the need for a region-specific development of diagnostic tools that can aid diagnosis and treatment in local context and prevention and limitation of pandemics in global context. . The figure showing the current life expectancy at birth based on the region of birth for both sexes in 20174. i& ii )Difference in cause of death between low and high socio-demographic index countries4.
Ischemic heart disease Low back pain. ii) Countries with a high socio-demographic index. (i) Countries with a low socio-demographic index.
IVD in developing countries
- Healthcare infrastructure in developing countries
- Extreme point of care testing need
Urban population distribution comprising 70% of healthcare infrastructure is located and accessible to 25-30% of the population, while the disease and disease prone rural population comprising ~70% relies on 20% of peripheral areas. centers (primary health center and non-clinical setting) for first-line treatment. In many rural areas, treatment diagnosis is neglected simply because it does not exist. Some of the key challenges include sample type, sample volume, number of samples, device footprint, power supply, ease of operation, time to result, cost, and data storage.
The strict quality criteria specific to each of these environments are regulated by the WHO, as almost 68% of countries in these regions do not have regulations23.
Lab-on-a-chip in different settings
The pressure-driven system, on the other hand, can perform complex analyzes in a single device, but requires complex instrumentation and a trained operator. Similar problems exist with electro-kinetic and acoustic systems, however integration and automation, centrifugal microfluidic systems, are relatively simple instrumentation and operation without complexity. The sample solution can flow from the sample pad to the absorbent pad by capillary-driven pumping18 (B) Continuous flow cell separation research using dielectrophoresis (DEP), the flow was driven by electrokinetics108 (C) Acoustic actuation of droplet movement, modulation of surface acoustic waves can transport droplets36 (D) Pico liter-scale liquid manipulation using microsyringes equipped directly on a chip19 (E) a lab-on-a-disk, sample solution and reagents can be transferred by centrifugal pumping38.
Centrifugal microfluidics
- General physics
- Centrifugal pumping
- Centrifugal microfluidic in IVD
- Instrumentation and operation of centrifugal microfluidic systems
- Clinical applications
- Clinical chemistry
- Immunoassays
- Nucleic acid tests
- Centrifugal microfluidic for EPOCT
- Opportunities of centrifugal microfluidics for EPOCT
- Disc Requirements
- Supporting Instruments
- Hand powered centrifuge for EPOCT
The average flow velocity (U) in a microchannel can be derived from the parameters; L is the length of the liquid in the microchannel, 𝑟̅ is the average distance of the liquid in the channels from the center of the disk, ∆𝑟 is the radial extent of the liquid.37. By rotating a disc, a centrifugal force is generated to induce radial transport of fluids from the center towards the outer edge of the disc. The dimensions of the channel associated with the speed of rotation, the location of the fluid chamber and the viscosity of the fluid determine the speed of flow in the channels.
Clinical chemistry involves relatively simpler assays than other IVD assays, and microfluidic operations can be fully automated for the entire assay simply by manipulating the inherent microgeometry of the disc. Figure 1.7 E and F shows some recent research on the integration of multiplex pathogen nucleic acid analysis and droplet PCR and on disk for sensitive detection47,48. A simplification and targeted approach to disk design and fabrication techniques would further reduce total system costs.
Affordability is a broad term used in most literature to specify the cost of the instrument. Most of the EPOCT setups do not have uninterruptible power supply and therefore the instrumentation design must be such that it can be powered without an external power source or solar power. The instrumentation must be self-sustaining in terms of power due to the lack of reliable power supply in EPOCT settings; therefore, devices that are powered by batteries and consume little power are preferred.
The detection technique should be chosen to exploit the use of the instrument for the widest possible range of biological applications. Some selective examples of the device are shown in Figure 1.8;55-59 Some of which have been tested with biological fluids such as blood and urine.
Design and characterization of diagnostic fidget spinner (Dx-FS)
- Abstract
- Introduction
- Designing Diagnostic fidget spinner (Dx-FS)
- Dx-FS design
- Fabrication of Dx-FS
- Working principles of Dx-FS with fluids and particles
- Choice of membrane
- Spin characterization
- Enrichment of bacteria with a few spins
- Characterization of the filtration rate of Dx-FS
- Introducing Fluid-assisted separation technology (FAST) in Dx-FS
- Comparing flowrates with FAST in Dx-FS
- Understanding operator variation while using Dx-FS
- Bacterial cell enrichment in Dx-FS
The red colored areas indicate the liquid flow during the process. f) Angular rotation frequency ω of Dx-FS versus time. When the device is set to rotate, the sample (colored in red) in the filling chamber experiences a centrifugal force (Figure 2.1e-1) that pushes the fluid through the membrane (Figure 2.1e-2). After identifying the device design and components, we set out to understand how the fluid will behave in motion inside the Dx-FS.
First, we set out to understand the spin rate profile of Dx-FS was quantified using the homemade setup shown in Figure 2.3a. The Dx-FS was installed in the light path and blocked the light during each spin. We also captured the detailed rotational motion of the Dx-FS using a high-speed camera (Phantom Miro 310; Vision Research, NJ, USA).
From the initial spin of Dx-FS, the angular rotation frequency reached its maximum and decreased with time (Figure 2.1f). Similarly, the total flow volume of a single spin was estimated from the measured angular rotation frequency of Dx-FS (Figure 2.4c). All the urine is removed by spinning by hand and bacterial cells remain on the membrane (Figure 2.4b).
The inlets (yellow) and vents (white) of the Dx-FS were incorporated with latex rings that prevent backflow or leakage during device operation. Bacterial cells are at a 99.35% recovery (Figure 3.1c), therefore, Dx-FS makes the concentration 126.5 times higher than the input urine sample (right panel).
Dx-FS for EPOCT/ POCT urinary tract infection diagnosis
Urinary tract infection diagnosis using Dx-FS
- Spiked bacteria isolation using Dx-FS
- Bacterial cell detection from fluorescence microscopy images
- Bacterial cell detection using colorimetric assay in Dx-FS
- Colorimetric detection of UTI Dx-FS
- Evaluating mammalian cells in urine interference in colorimetric assay
- Quantitative analysis of Dx-FS images
- Evaluating the effect of temperature and color stability
- Other detection methods integrated
- Pathogen identification using recombinase polymerase amplification (RPA)
- Gold nanoparticles (Au-NP)-based immunoassay in Dx-FS
Briefly, 100 μL detection solution (20 μL WST-8 diluted in 80 μL distilled water) was added to Dx-FS and incubated. Bacterial cell detection assay with (Dx-FS) and without (tube) enrichment stage. a) Samples of 1 ml with 106 bacterial cells/ml were stored in a tube or treated with Dx-FS. The bacterial cell detection assay was performed in a test tube (top) or Dx-FS (bottom). b) Optical density (OD) measurements of the detection test solution with samples of 102-105 CFU/mL in a tube or Dx-FS.
To quantify the same optical density measurement of both Dx-FS and the tube was measured and plotted in Figure 3.5b. The above cells were sprayed together with bacterial cells and the test was performed (Figure 3.6). The optical density (OD) of the solution was measured with a plate reader, NanoQuant Plate™, for nanodroplet measurements (Infinite 200 PRO, Tecan, Switzerland). Many previous reports have already demonstrated the use of hand warmer.93 As a potential alternative for such cases, a Dx-FS bacterial cell detection test was performed using a commercial hand warmer (Figure 3.8d).
Thermal map images of Dx-FS were acquired using a thermal imaging camera (Ti300 PRO, Fluke, USA). -FS bacterial cell detection assays were performed in an incubator or using a commercial hand warmer. a) Temperature of the Dx-FS membrane area measured using a thermal imaging camera. The bacteria were first enriched on the membrane in the Dx-FS device as described in Figure 3.1a (step 1 and step 2).
As shown in Figure 3.7, the LFA strip is inserted into the Dx-FS membrane area to absorb the amplified samples for 1 minute and then transferred to the buffer. After a one-hour incubation, the solution was introduced into Dx-FS and antibody-bound E.
Application of Dx-FS in POCT and EPOCT settings
- UTI diagnosis using patient sample
- Field trials at Kauvery Hospitals in India
- Routine Urine culture test of patient sample
- Outcomes of the study patient sample
- Antimicrobial susceptibility test (AST)
- Method of adapting AST on a fidget
- Fidget based antimicrobial susceptibility test (Fidget-AST)
- Clinical bacterial isolates for AST
- Antimicrobial selection and preparations
- Reference AST: Broth microdilution (BMD) test
The Dx-FS meets the standards provided by the WHO16 for ideal on-site testing in resource-constrained settings. In addition, Dx-FS was able to detect two cases that were false negative in the culture method. Proof of concept testing using Dx-FS. a) Urine samples of suspected UTIs tested by culture or Dx-FS.
The number of bacterial cells measured by cultivation or by Dx-FS is shown in the heat map. Dx-FS results are highly comparable to conventional culture-based UTI diagnosis in terms of sensitivity and specificity. The results confirm that UTI diagnosis by culture and Dx-FS were comparable based on ROC curves (Figure 4.2b).
Dx-FS provided a robust and versatile UTI diagnosis independent of sex, age and strains of bacterial cells (Figure 4.3). After the incubation, 1 ml of a sample was transferred to the Dx-FS sample chamber and enriched on the membrane by spinning. If the viable bacteria signal measured by Dx-FS for the antibiotic-treated sample is significantly reduced, it indicates that these bacteria are susceptible to the antibiotic drug.
The WST-8 colorimetric assay in Dx-FS measures the dehydrogenase activities of bacterial cells, which are closely related to the metabolic activity of the cells. Antimicrobial susceptibility testing of clinical isolates using Dx-FS. a) Normal AST takes at least two days for cell culture and AST; however, Fidget-AST takes less than 120 minutes to screen for phenotypic resistance.
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In the previous session we described the Dx fidget spinner device for the isolation of 60 bacteria. The figure shows the simple four-step approach that always compliments any porous material within 10 minutes. It has been well fabricated using our method, its scanning electron microscopic image, and the isolation of bacteria in the well has been imaged using a fluorescent microscope.
