PMID- 35448297
OWN - NLM
STAT- MEDLINE
DCOM- 20220425
LR  - 20220716
IS  - 2079-6374 (Electronic)
IS  - 2079-6374 (Linking)
VI  - 12
IP  - 4
DP  - 2022 Apr 12
TI  - Microfluidic One-Pot Digital Droplet FISH Using LNA/DNA Molecular Beacons for 
      Bacteria Detection and Absolute Quantification.
LID - 10.3390/bios12040237 [doi]
LID - 237
AB  - We demonstrate detection and quantification of bacterial load with a novel 
      microfluidic one-pot wash-free fluorescence in situ hybridization (FISH) assay in 
      droplets. The method offers minimal manual workload by only requiring mixing of 
      the sample with reagents and loading it into a microfluidic cartridge. By 
      centrifugal microfluidic step emulsification, our method partitioned the sample 
      into 210 pL (73 microm in diameter) droplets for bacterial encapsulation followed by 
      in situ permeabilization, hybridization, and signal detection. Employing locked 
      nucleic acid (LNA)/DNA molecular beacons (LNA/DNA MBs) and NaCl-urea based 
      hybridization buffer, the assay was characterized with Escherichia coli, 
      Klebsiella pneumonia, and Proteus mirabilis. The assay performed with single-cell 
      sensitivity, a 4-log dynamic range from a lower limit of quantification (LLOQ) at 
      ~3 x 10(3) bacteria/mL to an upper limit of quantification (ULOQ) at ~3 x 10(7) 
      bacteria/mL, anda linearity R(2) = 0.976. The total time-to-results for detection 
      and quantification was around 1.5 hours.
FAU - Kao, Yu-Ting
AU  - Kao YT
AUID- ORCID: 0000-0003-4671-7907
AD  - Laboratory for MEMS Applications, IMTEK-Department of Microsystems Engineering, 
      University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
AD  - Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 
      01-224 Warsaw, Poland.
FAU - Calabrese, Silvia
AU  - Calabrese S
AD  - Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
FAU - Borst, Nadine
AU  - Borst N
AUID- ORCID: 0000-0003-1258-3250
AD  - Laboratory for MEMS Applications, IMTEK-Department of Microsystems Engineering, 
      University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
AD  - Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
FAU - Lehnert, Michael
AU  - Lehnert M
AD  - Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
FAU - Lai, Yu-Kai
AU  - Lai YK
AUID- ORCID: 0000-0003-1037-9846
AD  - Laboratory for MEMS Applications, IMTEK-Department of Microsystems Engineering, 
      University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
AD  - Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 
      01-224 Warsaw, Poland.
AD  - Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
FAU - Schlenker, Franziska
AU  - Schlenker F
AUID- ORCID: 0000-0003-2082-1677
AD  - Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
FAU - Juelg, Peter
AU  - Juelg P
AD  - Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
FAU - Zengerle, Roland
AU  - Zengerle R
AD  - Laboratory for MEMS Applications, IMTEK-Department of Microsystems Engineering, 
      University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
AD  - Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
FAU - Garstecki, Piotr
AU  - Garstecki P
AUID- ORCID: 0000-0001-9101-7163
AD  - Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 
      01-224 Warsaw, Poland.
FAU - von Stetten, Felix
AU  - von Stetten F
AUID- ORCID: 0000-0002-8547-0495
AD  - Laboratory for MEMS Applications, IMTEK-Department of Microsystems Engineering, 
      University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
AD  - Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
LA  - eng
GR  - 711859/Marie Sklodowska-Curie/
PT  - Journal Article
DEP - 20220412
PL  - Switzerland
TA  - Biosensors (Basel)
JT  - Biosensors
JID - 101609191
RN  - 0 (Oligonucleotides)
RN  - 0 (locked nucleic acid)
RN  - 9007-49-2 (DNA)
SB  - IM
MH  - *DNA
MH  - Escherichia coli/genetics
MH  - In Situ Hybridization, Fluorescence/methods
MH  - *Microfluidics/methods
MH  - Oligonucleotides
PMC - PMC9032532
OTO - NOTNLM
OT  - amplification-free detection
OT  - digital assays
OT  - droplet microfluidics
OT  - fluorescence in situ hybridization
OT  - locked nucleic acid
OT  - molecular beacons
COIS- The authors declare no conflict of interest. The funders had no role in the 
      design of the study; in the collection, analyses, or interpretation of data; in 
      the writing of the manuscript, or in the decision to publish the results.
EDAT- 2022/04/22 06:00
MHDA- 2022/04/26 06:00
PMCR- 2022/04/12
CRDT- 2022/04/21 20:19
PHST- 2022/03/05 00:00 [received]
PHST- 2022/04/06 00:00 [revised]
PHST- 2022/04/10 00:00 [accepted]
PHST- 2022/04/21 20:19 [entrez]
PHST- 2022/04/22 06:00 [pubmed]
PHST- 2022/04/26 06:00 [medline]
PHST- 2022/04/12 00:00 [pmc-release]
AID - bios12040237 [pii]
AID - biosensors-12-00237 [pii]
AID - 10.3390/bios12040237 [doi]
PST - epublish
SO  - Biosensors (Basel). 2022 Apr 12;12(4):237. doi: 10.3390/bios12040237.