88
L. Acosta-Soto and S. Hosseini
this strategy, the device was tested with fluorescent dye Lucifer yellow in presence of
the emission filter Chroma 59,004 with transmission windows at both 500–535 nm
and 570–620 nm resulting in satisfactory imaging and detection of the two windows.
The smaller range corresponds to the green emission and the larger one to the red
emission. An additional reason that this setup is a viable option for large-scale cells
flow cytometry is the optimization of signal to the background and the source noise.
The utilization of micro-ball lenses has improved this ratio 18 times against detection
the systems without micro-ball lenses.
The objective of flow cytometry studies is usually to study live cells, rather than
dyes. Fan et al. (2013) achieved detection of two cells (HeLa and Ramos cells) with
different fluorescent tags along with accurate readout of respective proportions in
the sample supplied to the device. Multiple and simultaneous imaging, however, is
a demanding process, particularly when large volumes and fast results are needed.
In any case, the sample must be initially tagged, which is not a simple task when the
analyte is a strand of a genetic material.
One of the applications of LOC devices is the detection of antibiotic resistance in sepsis-related scenarios (Knob et al. 2018). In this case, specific DNA
strands must be located and tagged for further analysis of a sample. This preparation requires the lysis of the sample bacteria cells, obtained through prior separation from the patient’s cells. Once the genetic material is available, it is possible
to employ various strategies to bind molecular markers to a given strand. Recently,
a monolith within a polypropylene (PP) was used in a microfluidic device with
specific functionalization to trap DNA sequences. In their study, Knob et al. (2018)
immobilized genes related to Klebsiella pneumoniae carbapenemase (KPC). This
was used as a mechanism to analyze the bacterial resistance to carbapenems
by exposing extracted DNA to a monolith fabricated from crosslinkers exclusively. These crosslinkers were polyethylene-glycol diacrylate (PEGDA) and 3,4Ethylenedioxy-N-methylamphetamine (MDMHA) functionalized with a tailored 90mer specific to the DNA of interest. Once captured by the monolith, the genetic
material was then exposed to hybridization probes designed as molecular beacons to
bind the fluorophore to the target. For this step, the sequence needed to bind to the
KPC genes was determined through software simulations and analysis. The study
showed that a modified molecular beacon that has two fluorophores rather than just
one was able to effectively detect and signal the presence of the target DNA. This has
validated the use and functionality of the single-step fabricated monolith proposed
for capture, labeling, and eluting of analyte.
Our understanding of the efficiency of molecular probes and dyes as fluorescent emitters has led the advancements in the field to another class of fluorophores,
quantum dots (QD). QDs offer an alternative with higher photostability, availability,
and adaptability for specific applications. QDs were applied in the detection of carcinoma cell apoptosis (Montón et al. 2017), as described in Table 3.1. When cells enter
the state of apoptosis, it is common to observe the translocation of phosphatidylserine (Ps) from the inner layer of the cellular membrane to the exterior one. This
presents a unique possibility to identify this process of cellular death by tracking Ps
translocation through QD tagging. By binding a QD to the apoptotic cell, annexin V
L. Acosta-Soto and S. Hosseini
this strategy, the device was tested with fluorescent dye Lucifer yellow in presence of
the emission filter Chroma 59,004 with transmission windows at both 500–535 nm
and 570–620 nm resulting in satisfactory imaging and detection of the two windows.
The smaller range corresponds to the green emission and the larger one to the red
emission. An additional reason that this setup is a viable option for large-scale cells
flow cytometry is the optimization of signal to the background and the source noise.
The utilization of micro-ball lenses has improved this ratio 18 times against detection
the systems without micro-ball lenses.
The objective of flow cytometry studies is usually to study live cells, rather than
dyes. Fan et al. (2013) achieved detection of two cells (HeLa and Ramos cells) with
different fluorescent tags along with accurate readout of respective proportions in
the sample supplied to the device. Multiple and simultaneous imaging, however, is
a demanding process, particularly when large volumes and fast results are needed.
In any case, the sample must be initially tagged, which is not a simple task when the
analyte is a strand of a genetic material.
One of the applications of LOC devices is the detection of antibiotic resistance in sepsis-related scenarios (Knob et al. 2018). In this case, specific DNA
strands must be located and tagged for further analysis of a sample. This preparation requires the lysis of the sample bacteria cells, obtained through prior separation from the patient’s cells. Once the genetic material is available, it is possible
to employ various strategies to bind molecular markers to a given strand. Recently,
a monolith within a polypropylene (PP) was used in a microfluidic device with
specific functionalization to trap DNA sequences. In their study, Knob et al. (2018)
immobilized genes related to Klebsiella pneumoniae carbapenemase (KPC). This
was used as a mechanism to analyze the bacterial resistance to carbapenems
by exposing extracted DNA to a monolith fabricated from crosslinkers exclusively. These crosslinkers were polyethylene-glycol diacrylate (PEGDA) and 3,4Ethylenedioxy-N-methylamphetamine (MDMHA) functionalized with a tailored 90mer specific to the DNA of interest. Once captured by the monolith, the genetic
material was then exposed to hybridization probes designed as molecular beacons to
bind the fluorophore to the target. For this step, the sequence needed to bind to the
KPC genes was determined through software simulations and analysis. The study
showed that a modified molecular beacon that has two fluorophores rather than just
one was able to effectively detect and signal the presence of the target DNA. This has
validated the use and functionality of the single-step fabricated monolith proposed
for capture, labeling, and eluting of analyte.
Our understanding of the efficiency of molecular probes and dyes as fluorescent emitters has led the advancements in the field to another class of fluorophores,
quantum dots (QD). QDs offer an alternative with higher photostability, availability,
and adaptability for specific applications. QDs were applied in the detection of carcinoma cell apoptosis (Montón et al. 2017), as described in Table 3.1. When cells enter
the state of apoptosis, it is common to observe the translocation of phosphatidylserine (Ps) from the inner layer of the cellular membrane to the exterior one. This
presents a unique possibility to identify this process of cellular death by tracking Ps
translocation through QD tagging. By binding a QD to the apoptotic cell, annexin V