| 1. |
- Dietvorst, Jiri, et al.
(författare)
-
Bacteria Detection at a Single-Cell Level through a Cyanotype-Based Photochemical Reaction
- 2022
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 94:2, s. 787-792
-
Tidskriftsartikel (refereegranskat)abstract
- The detection of living organisms at very low concentrations is necessary for the early diagnosis of bacterial infections, but it is still challenging as there is a need for signal amplification. Cell culture, nucleic acid amplification, or nano-structure-based signal enhancement are the most common amplification methods, relying on long, tedious, complex, or expensive procedures. Here, we present a cyanotype-based photochemical amplification reaction enabling the detection of low bacterial concentrations up to a single-cell level. Photocatalysis is induced with visible light and requires bacterial metabolism of iron-based compounds to produce Prussian Blue. Bacterial activity is thus detected through the formation of an observable blue precipitate within 3 h of the reaction, which corresponds to the concentration of living organisms. The short time-to-result and simplicity of the reaction are expected to strongly impact the clinical diagnosis of infectious diseases.
|
|
| 2. |
|
|
| 3. |
- Iyengar, Sharath Narayana, et al.
(författare)
-
High resolution and rapid separation of bacteria from blood using elasto‐inertial microfluidics
- 2021
-
Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 42:23, s. 2538-2551
-
Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Improved sample preparation has the potential to address unmet needs for fast turnaroundsepsis tests. In this work, we report elasto-inertial based rapid bacteria separation from diluted blood at high separation efficiency. In viscoelastic flows, we demonstrate novel findings where blood cells prepositioned at the outer wall entering a spiral device remain fullyfocused throughout the channel length while smaller bacteria migrate to the opposite wall.Initially, using microparticles, we show that particles above a certain size cut-off remainfully focused at the outer wall while smaller particles differentially migrate toward the inner wall. We demonstrate particle separation at 1 μm resolution at a total throughput of1 mL/min. For blood-based experiments, a minimum of 1:2 dilution was necessary to fullyfocus blood cells at the outer wall. Finally, Escherichia coli spiked in diluted blood were continuously separated at a total flow rate of 1 mL/min, with efficiencies between 82 and 90%depending on the blood dilution. Using a single spiral, it takes 40 min to process 1 mLof blood at a separation efficiency of 82%. The label-free, passive, and rapid bacteria isolation method has a great potential for speeding up downstream phenotypic and genotypicanalysis.
|
|
| 4. |
- Iyengar, Sharath Narayana, et al.
(författare)
-
High throughput separation of bacteria from blood for sepsis diagnostics using extended elasto-inertial microfluidics
- 2019
-
Ingår i: 23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2019. - : Chemical and Biological Microsystems Society. ; , s. 590-591
-
Konferensbidrag (refereegranskat)abstract
- Separation of bacteria from blood for sepsis diagnostics has received substantial interest due to lack of high throughput alternatives. Here, we introduce extended elasto-inertial microfluidics based high throughput (1 mL/min) separation of bacteria from whole blood. We demonstrate separation of E.coli from 1 mL of whole blood in 40 min using a single spiral chip with 90% separation efficiency. This opens up opportunities by aiding for downstream analysis, by reducing the time of sample preparation for sepsis diagnosis.
|
|
| 5. |
|
|
| 6. |
|
|
| 7. |
- Iyengar, Sharath Narayana
(författare)
-
Novel microfluidic based sample preparation methods for rapid separation and detection of viable bacteria from blood for sepsis diagnostics
- 2021
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Sepsis is a serious medical condition characterized by a whole-body inflammatory response caused by bloodstream infection. The final stage of sepsis can lead to septic shock, multiple organ failure, and death. In early sepsis, the concentration of bacteria in the bloodstream is typically low, making diagnosis challenging. Rapid diagnosis of sepsis is crucial as there is an exponential increase in mortality for every hour delay in the appropriate antibiotics administration. Common culture-based methods fail in fast bacteria determination as it takes up to 24-72 hr. On the other hand, recent rapid nucleic acid-based diagnostic methods are prone to false positives from human DNA mainly due to a lack of efficient sample preparation methods. This Ph.D. work was aimed at the development of novel sample preparation methods for rapid and efficient separation and identification of bacteria from blood for sepsis diagnostics. To address this, two different approaches were explored. In the first approach, a label-free, size-based, passive elasto-inertial microfluidics (visco-elastic flows) method was developed (Paper I and II). Initially, behavior of particles were studied in solution containing polyethylene oxide (PEO) using different spiral designs (Paper I). By using the knowledge from paper I, a spiral design was used to preposition the particles at the outer wall of the inlet using PEO as sheath and we showed that a particle of a certain size remains fully focused at the outer wall throughout the channel length. The optimized parameters were extended to demonstrate that when bacteria is spiked into diluted blood, blood remains fully focused at the outer wall throughout the channel length while smaller bacteria differentially migrate towards the inner wall for rapid separation. Using E.coli spiked into the diluted blood sample, bacteria separation is demonstrated at an efficiency of 82 to 90% depending on the blood dilution using a single spiral chip (Paper-II). The second approach (Paper III) involves a selective cell lysis method where lysis buffer composition is optimized to selectively lyse blood cells in 5 min while maintaining bacterial viability. The lysed blood cells were filtered through a filter paper to capture viable bacteria. The captured bacteria on the filter paper was detected using Prussian blue (PB) colorimetric analysis. In PB color-based assay, viable bacteria metabolically reduce iron (III) complexes, initiating a photo-catalytic cascade toward PB formation on the filter paper visible to the naked eye. Using this approach it was possible to detect bacteria by the naked eye. This approach was also further optimized to perform antibiotic susceptibility testing to determine the minimum inhibitory concentration (MIC). Furthermore, as a step towards rapid genomic analysis, a novel method combining ITP-RCA (Isotachophoresis – Rolling Circle Amplification) was studied and optimized for real-time amplification (RCA), focusing and detection of bacterial DNA in a microfluidic channel (Paper IV). In this study we demonstrate rapid and increased sensitivity of bacterial DNA detection. This method has a huge potential to accelerate the time needed for DNA based analysis for infectious diseases. All in all, the ability of these sample preparation methods for rapid and effective separation and detection of key pathogens in blood will help in decreasing the time of sepsis diagnosis and aid towards efficient phenotypic or genotypic analysis.
|
|
| 8. |
- Iyengar, Sharath Narayana, et al.
(författare)
-
Rapid detection of viable bacteria in whole blood for early sepsis diagnosis and suseptibility testing
- 2021
-
Ingår i: Proceedings MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - : Chemical and Biological Microsystems Society. ; , s. 791-792
-
Konferensbidrag (refereegranskat)abstract
- Sepsis is a serious medical condition characterized by a whole-body inflammatory state caused by infection. Here, we present a sepsis method for rapid detection of bacteria from whole blood in less than 5h, combining selective blood cell lysis and a sensitive colorimetric based detection method. Selective cell lysis buffer allows selective rupture of blood cells (5 min), while maintaining bacteria 100% viable. Viable bacteria metabolically reduce iron (III) complexes, initiating a photo-catalytic cascade toward Prussian Blue formation visible to the naked eye. The method is finally validated for antibiotic susceptibility testing.
|
|
| 9. |
- Iyengar, Sharath Narayana, et al.
(författare)
-
Toward Rapid Detection of Viable Bacteria in Whole Blood for Early Sepsis Diagnostics and Susceptibility Testing
- 2021
-
Ingår i: ACS Sensors. - : American Chemical Society (ACS). - 2379-3694. ; 6:9, s. 3357-3366
-
Tidskriftsartikel (refereegranskat)abstract
- Sepsis is a serious bloodstream infection where the immunity of the host body is compromised, leading to organ failure and death of the patient. In early sepsis, the concentration of bacteria is very low and the time of diagnosis is very critical since mortality increases exponentially with every hour after infection. Common culture-based methods fail in fast bacteria determination, while recent rapid diagnostic methods are expensive and prone to false positives. In this work, we present a sepsis kit for fast detection of bacteria in whole blood, here achieved by combining selective cell lysis and a sensitive colorimetric approach detecting as low as 10(3) CFU/mL bacteria in less than 5 h. Homemade selective cell lysis buffer (combination of saponin and sodium cholate) allows fast processing of whole blood in 5 min while maintaining bacteria alive (100% viability). After filtration, retained bacteria on filter paper are incubated under constant illumination with the electrochromic precursors, i.e., ferricyanide and ferric ammonium citrate. Viable bacteria metabolically reduce iron(III) complexes, initiating a photocatalytic cascade toward Prussian blue formation. As a proof of concept, we combine this method with antibiotic susceptibility testing to determine the minimum inhibitory concentration (MIC) using two antibiotics (ampicillin and gentamicin). Although this kit is used to demonstrate its applicability to sepsis, this approach is expected to impact other key sectors such as hygiene evaluation, microbial contaminated food/beverage, or UTI, among others.
|
|
| 10. |
- Kumar, Tharagan, et al.
(författare)
-
High throughput viscoelastic particle focusing and separation in spiral microchannels
- 2021
-
Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 11:1
-
Tidskriftsartikel (refereegranskat)abstract
- Passive particle manipulation using inertial and elasto-inertial microfluidics have received substantial interest in recent years and have found various applications in high throughput particle sorting and separation. For separation applications, elasto-inertial microfluidics has thus far been applied at substantial lower flow rates as compared to inertial microfluidics. In this work, we explore viscoelastic particle focusing and separation in spiral channels at two orders of magnitude higher Reynolds numbers than previously reported. We show that the balance between dominant inertial lift force, dean drag force and elastic force enables stable 3D particle focusing at dynamically high Reynolds numbers. Using a two-turn spiral, we show that particles, initially pinched towards the inner wall using an elasticity enhancer, PEO (polyethylene oxide), as sheath migrate towards the outer wall strictly based on size and can be effectively separated with high precision. As a proof of principle for high resolution particle separation, 15 mu m particles were effectively separated from 10 mu m particles. A separation efficiency of 98% for the 10 mu m and 97% for the 15 mu m particles was achieved. Furthermore, we demonstrate sheath-less, high throughput, separation using a novel integrated two-spiral device and achieved a separation efficiency of 89% for the 10 mu m and 99% for the 15 mu m particles at a sample flow rate of 1 mL/min-a throughput previously only reported for inertial microfluidics. We anticipate the ability to precisely control particles in 3D at extremely high flow rates will open up several applications, including the development of ultra-high throughput microflow cytometers and high-resolution separation of rare cells for point of care diagnostics.
|
|
