chem test 1 paper notes Flashcards

fluorescence tech widely used bc of simple operation, high sensitivity, good selectivity, high spatial / temporal resolution

traditional fluorophores often suffer from aggregation caused quenching (ACQ) at high concentrations or in aggregated state (weakened luminescence)

too much emission in dilute solutions, because t strong fluorescence in aqueous soln bc of poor water solubility (since not water soluble, they aggregate which = fluorescence prematurely)

class compounds - aggregation induced emission (AIE) - bc were opposite in that showed negligible emission in dilute solns, but enhanced fuorescence in aggregate / solid state

• AIE mechanism: restriction of intramolecuar motion (RIM) played key role in AIE phenomenon

since then, AIEgens developed for many things

thought process: AIEs seem great!!! RIGHT? like wow discovered a new / better version of what had before (ACQ). are there drawbacks??? SEE NEXT CARD

• suffer from complex organic synthesis, high cost of environmental damage, potential biological toxicity
• can also emit strong fluorescence in aqueous soln bc of poor water solubility
  • but we need them to NOT emit in aq solns.

lead to: therefore -> must explore new environmentally friendly, biocompatible, water-soluble AIEgens

...

could be solution?

• jatrorhizine (Jat) chloride - from chinese herb
  • found to be a natural water soluble AIEgen not previously reported

• not emit fluorescence in aq. soln even containing DNA/polymers (AMAZING, WHAT THEY WANTED TO SOLVE) until the formation of DNA hydrogel
  • bc of good water solubility/AIE character
  • found when they did DNA-based assay of miRNA to confirm its ability for fluoresncece biosensing in aq. soln

miRNA is closely associated w/ diseases. need biomarker detection at low concentration (routine (standard) detection methods are not sensitive enough to detect them)

• DNA hydrogen fluorescence biosensor based on Jat chloride and EDC designed for ultrasensitive + label-free detection of miRNA

1. entropy driven signal amplification
   • DNA output + template mixed = substrate
   • -> target (miRNA) = catalyst to trigger 1st chain displacement reaction + stimulate substrate to release outputs
   • fuel DNA triggers the second chain displacement rxn + compels target and another output to be released
   • = each cycle release 2 outputs
   • = trigger part 2
2. released outputs -> opening of 2 hairpin DNAs + prompt 2 copolymer chains to form DNA hydrogel
   • = strong fluorescence produced (emission) from Jat chloride is in the hydrogen, restricted
   • = biosensing strategy can realize the label-free detection of miRNA with low limit of detection

this is reliable and stable. acheives ultrasensitive, label free detection of miRNA by combining EDC, AIEgens, and DNA hydrogels + expands application of water-soluble AIEgens and natural AIEgens

...

• output + template incubated in Tris-HCl buffer = get substrate
• targets + fuel added to substrate soln
• incubated = trigger EDC process
• = large # outputs (2^n magnification) released after n cycles of subsequent gel-forming process

• ammonium persulfate (APS) and N,N,N',N'-tetramethylethylenediamine (TEMED)
• polyacrylamide DNA modified chains synthesized according to literature
  • HEPEs buffer soln for H1 and H2
  • TEMED added
• incubated = copolymer chains P1/P2 to obtain uniform/stable DNA hairpin structures on copolymer chains
• = prepared polymer chains products

• Jat soln added to mixture of P1 and P2, entropy driven amp products, and final volume adjusted to 300 microL with HEPES buffer
• mixed soln -> 96 well plates + incubated = DNA hydrogels
• fluorescnece spectra of the hydrogels collected by mircopla`te reader

...

• Jat available + natural molecule
  • excitation peaks 355 and 450 nm in THF
  • = chose 355 as excitation wavelength bc was high excitation than 450
  • emission peak of Jat =550 nm
  • AIE characteristics studied by investigating photoluminescence (PL) behavior in water and water/THF mixtures

• Jat: almost no emission in diluate aq. solns. Enhances emission with inc of the THF fraction
• Jat in water/THF mixtures in UV lamp also proved enhanced emission w/ inc in aggregation degree = Jat is an AIE-active molecule
• enhacned emission in water/THF mixture attributed to weak solubility + aggregation in weakly polar solvents = shoed AIE mechanism of Jat chloride is RIM

Jat in THF/water mixture was analyzed using dynamic light scattering (DLS)

• when THF fraction is 10%, small sizes of aggregates formed bc of inslolubility of Jat in THF
• aggregate size inc if more THF
• THF fraction 99%, slightly reduced aggregate size bc of hydration shell in hydropenic environment
• = aggregations in weakly polar solvents

Jat fluoresence in diff polarities was observed

• polarity soln decrease, fluoresence intensity increased =. suggest AIE effect of Jat in weakly polar solvents

photoluminescence quantum yields (PLQYs) of dispersed Jat in water, nanosuppression, powder were 0.31% (relative std dev = 5.04%), 1.23% (RSD = 4.72%), and 3.37% (RSD = 4.01%) = suggest Jat was AIE-active

further show AIE character: measured emission lifetimes

• 2.72 and 3.65 ns in nannosuppresion and powder
• 0.41 ns in water

• crystal structure determined to better understand the photophysical behavior of Jat
  • crystal Xray diffraction
  • crystallized into monolytic needle like crystals
  • main moiety adopts nonplanar conformation in its crystal with dihedral angle of ...
  • torsion angles... suggest possible existence of twisted IM charge transfer (TICT) effect _ IM vibronic motion behavior
  • IM and intermolecular H bonds rigidify molecular conformation and intensify fluorescent emission of Jat chloride in crystal state
• weak fluorescence of Jat chloride in dilute aq. solns may be due to TICT effect and active IM motion which lead to nonradiative decay
• in aggregate or solid state, nonradiative decay pathways is prominently restricted, resulting in strong fluorescence

• reveal AIE mechanism of Jat - IM motion and TICT studied
  • showed active IM motion + weak fluorescence in dilute aq. solns
  • showed restricted IM motion + bright fluorescence in aggregate state
  • diff polarities of solvents studied
    • UV-vis absorption of Jat almost unchanged, but fluoresence intensity DEC with INC in solvent polarity
    • = indicate that TICT effects exist in Jat molecules
      • TICT = photophysical phenomenon of donor-acceptor conjugated small molecules. When molecules excited in polar solvent -> turn from excited to TICT state + from TICT to ground state can = nonradiative decay + then fluorescence quenching (any process that decreases the fluorescence intensity of a sample)
      • jat = typical example of donor/acceptor structure = fluorescence intensity of Jat decreased obviously in polar solvents
      • summary:
      • HOMO and LUMO explored to understand mechanism of AIE-active Jat

• investigate potential applications of Jat - ultrasensitive DNA hydrogel fluorescent biosensor made (to detect microRNA)
  • determined entropy driven signal amplification confirmed by PAGE
    • bands (qualitative)
    • showed large # outputs produced by entropy driven signal amp process
    • outputs can initiate hybridization chain reaction (HCR) to generate high molec weight products in mixture of H1 and H2
• examined gel formation of P1, mixture of P1/P2, mixture of P1, P2, and products of entropy driven amp (outputs) in natural light and UV rad
  • results = indicate DNA hydrogel formed only in presence of outputs
    • fluoresence intensity sig increased upon formation of DNA hydrogel (could also tell under UV light)
    • change in fluorescent intensity proportional to output #, which were related to concentration of microRNA
    • = DETERMINATION OF microRNA COULD BE REALIZED BY DETECTION OF FLUORESCENCE INTENSITY OF DNA HYDROGEL

fluorescense emission comes from aggregated Jat in DNA hydrogel, so [Jat] was optimized first

• best signal to noise ratio at 40 microM of Jat

# outputs from entropy signal amp influenced fluorescence intensity a lot

• optimized this
• inc fuel + substrate = fl intensity increase + became stable when conc. of fuel and substrate exceeded 0.8 and 2.5 microM respectively and at 80 min. used this

time for formation of DNA hydrogel optimized

• fluor intensity inc when inc in incubation time until 120 mins -> used this

to investiagte sensitivity + quantitative application of biosensor designed, diff conc. of miRNA-21 monitored by proposed biosensor under optimal conditions

• fluor intensity inc w/ miRNA inc (0-100nM)
• LINEAR relationship btw fluorescence intensity and log miRNA-21 conc. (1 fM-100nM)
  • linear regression Y (fluor intensity) = 182.6 + 55.4lgc (log of miRNA conc.) with correlation coef R^2 = 0.996

LIMIT OF DETECTION (LOD) - ESTIMATED TO BE 0.049 fM FOR MIRNA-21 DETECTION. LOWER THAN PREVIOUS ASSAY METHODS REPORTED

RESULT = HIGHLY SENSITIVE MIRNA ASSAY CAN BE REALIZED BY MONITORING CHANGE IN FLUORESCENCE INTENSITY OF SYSTEM

fluo biosensor proposed = versatile assay platform -> can detect other targets by changing DNA base sequence of template, outputs, fuels

antiinterference capability of strategy to detect miRNA-21, used 4 analogs that mixed w/ target (miRNA-21)

• RESULT = analogs hardly interfered w/ determination of target

stability of assay investigated

• fluo intensity relatively stable

show practicability of assay

• miRNA-21 in human serum detected using STANDARD ADDITION METHOD - ADDED KNOWN CON. MIRNA-21 TO 100FOLD DILUTED HUMAN SERUM + DETERMINE CONC. MIRNA-21 IN SPIKED SAMPLES
  • RECOVERY RANGED 99.8-101.15 (4.3-5.1%)
  • showed DNA hydrogen fluorescent biosensor showed good antiinterference capability in complex matrix

• Jat chloride = novel AIEgen
  • investigated AIE property + mechanism
  • predict AIE phenomenon caused by RIM and TICT effects (see via data)
  • good water solubility compared to other AIEgens = facilitated bioassay in aq. solns even w/ dna and polymers
  • could be used as fluorescent probe to make DNA hydrogel fluorescent biosensor for detection of trace microRNA
  • TO INCREASE DETECTION SENSITIVITY, ENTROPY DRIVEN SIGNAL AMP AND DNA HYDROGEL INDUCED EMISSION OF JAT WERE APPLIED SUCCESSFULLY
  • proposed DNA hydrogel fluorescent biosensor showed good sensitivity, good selectivity, high stability, excelent renewability, and acceptable reliability for a real sample assay

...

microRNA (miRNA).

The DNA hydrogel fluorescence biosensor developed in the paper uses miRNA as a catalyst to trigger the entropy-driven circuit, which leads to the aggregation of Jat and the turn-on of fluorescence. Therefore, the biosensor can be used to detect miRNA in a label-free and ultrasensitive manner.

this miRNA assay was used to confirm Jat's superior fluorescence biosensing in aqueous solution.

miRNA = biomarker

novel DNA hydrogel fluorescence biosensor based on a water-soluble natural AIEgen, jatrorrhizine (Jat), for ultrasensitive and label-free detection of miRNA.

potential to be used for early diagnosis + monitoring of diseases

DEVICE is quantitative because it can be used to measure the concentration of miRNA in a sample.

ex:

The DNA hydrogel fluorescence biosensor has an ultralow limit of detection (0.049 fM, S/N = 3) for miRNA. It also has excellent stability and acceptable reliability for real sample assay.

in the study overall, researchers used PAGE (qualitative) to determine if amplification was working

used quantitative and qualitative measurements to determine AIE characteristics of Jat (ex: structure, SEM, intensity with diff amounts THF, etc.)

novel DNA hydrogel fluorescence biosensor based on using Jat for detection of trace miRNA

need is that the fluorophore should not emit light in an aqueous solution, even containing DNA and other biomacro- molecules, until the target biomolecules are added.

important to explore new environmentally friendly, biocompatible, and water-soluble AIEgens.

it is water soluble and thus does not emit fluorescence in aqueous solution until the formation of DNA hydrogel, this makes it superior in fluorescence biosensing in aqueous solution. This is due to its IM motion and TICT which lead to nonradiative decay. in aggregate, nonradiative decay is restricted = strong fluorescence

major challenge that hinders biomarker detection includes: some biomarkers exist at extremely low concentrations, and the routine detection methods are not sensitive enough to detect them.

his was solved by successfully incorporating entropy-driven amplification, allowing the proposed device to be ultra sensitive

it acts as a fluorescent probe in the proposed device to facilitate detection of miRNA via DNA hydrogel and entrop driven amp

miRNA-21 in human serum was detected by using a standard addition method. A series of samples were prepared by adding known concentrations of miRNA-21 to 100-fold-diluted human serum and then the concentrations of miRNA-21 were determined in the spiked samples

spiked determine anti interference - determine if analogs to miRNA would affect results

all had about 100% recovery ish

Seems that since it went well there was no interferences with the complex matrix and the percent recovery values are really high which is good

further emphasizes the selectivity of the method.

yes

1. to test interference of miRNA assay w/ different analogs. in determining anti interference capability of the assay

limit of detection in this experiment tells lowest concentration or amount of analyte, miRNA, reliably detected by the assay. Says something about how well the assay can detect low concentrations of the analyte

To further verify the practicability of the assay system, miRNA-21 in human serum was detected by using a standard addition method. A series of samples were prepared by adding known concentrations of miRNA-21 to 100-fold-diluted human serum and then the concentrations of miRNA-21 were determined in the spiked samples

used standard addition to determine if analogs to miRNA-21 would interfere with results

A lower limit of detection of miRNA was achieved by employing entropy driven amplification, allowing the assay to have a low limit of detection (can detect low concentrations of the analyze, miRNA)

it restricts IM movement of Jat which = strong fluorescence emission

to synthesize the DNA modified polyacrylamide polymer chains

• good sensitivity
  • was able to detect lower concentrations of miRNA (analyte)
• good selectivity
  • little/no interference even by analogs
• stability
• renewability
  • nonexpensive + efficient
• reliability

two buffers used: Tris-HCl Buffer in the entropy-driven amplification reaction. Tris-HCl used to obtain substrate for EDC process

the HEPES buffer in the formation of the DNA hydrogel and fluorescence detection.

0.049 fM

• tested fluorescence of of Jat in solvents of different polarities
• (photoluminescence quantum yields) (PLQYs)
• = %5.04, 4.72, 3.37, showed

miRNA detection

EDC and DNA based amplification

dynamic light scattering (DLS) to prove that formation of aggregates occurs of Jat in weakly polar solvents

[Jat]

• best signal to noise ratio at 40 microM of Jat

# outputs ffrom EDC bc it influences fluorescence intensity a lot

• optimized this
• inc fuel + substrate = fl intensity increase + became stable when conc. of fuel and substrate exceeded 0.8 and 2.5 microM respectively and at 80 min. used this

time for formation of DNA hydrogel optimized

• fluor intensity inc when inc in incubation time until 120 mins -> used this

• photoluminescence behaviors in water and THF mixtures (QUANT)
  • almost no luminescence in dilute aq. solns
  • more emossion with inc THF fraction bc insoluble in THF -> makes hydration shell = aggregates = emission
  • indicated Jat is AIE active molecule
  • polarity dec = emission increase
• emission lifetimes determined
  • in good accordance with AIE characteristics of Jat
  • QUANT
• xray single-crystal X-ray diffraction
  • crystal structure determined
  • H bonds rigidify = stronger fluorescence
  • QUAL/QUANT?
• UV VIS absorption???
  
  • show AIE mechanism
  • more IM motion = less fluorescence and vice versa
• application of Jat
  • assay biosensor to detect miRNA

higher concentration of miRNA

PAGE

band 10 - positive control

1-5 negative controls

negative control - testing if DNA hydrogel form with everything except output to determine that output is necessary for hydrogel formation

• diff concentrations miRNA-21
  • intensity (QUANT inc w/ miRNA con from 0 - 100nM
  • linear btw intensity/log of miRNA-21
  • eq: Y = 182.6 + 55.41 lgc
• EDC successfully applied
• low interference
  • analogs hardly interfere
  • SELECTIVITY

yes, for the intensity of the fluorescence using the assay with miRNA

in using analogs along with the assay to determine anti-interference, which shows good selectivity

yes, if DNA base, template, and fuels are changed.

fluorescence intensity is stable (RSD = 3.72) for about 72 hrs

aggregation-induced emission luminogen

EDC, to increase signal and thus decrease the detection limit (detects lower/trace amounts of miRNA)

bc nucleic acid is a versatile construction material for biosensors bc of its predictability and programmability of watson-crick base pairing

1. EDC
   • miRNA = target and catalyst to trigger chain displacemet = substrate release outputs and fuel DNA triggers second one
   • = 2 outputs released
2. formation of DNA hydrogel
   • outputs trigger part 2
   • lead to opening of hairpin DNA (H1 and H2)
   • -> + prompt copolymer chains (P1 and P2) to form hydrogel, which restricts Jat and = stronger emission

is a weakly polar solvent, used to determine Jat's aggregation (emission) in more/less polar solvents to assess its AIE characteristics

diff concentrations miRNA-21

• intensity QUANT inc w/ miRNA con from 0 - 100nM
• and LOD is 0.049fM