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Literature Article Analysis: Fluorescence

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    Out-of-Class Questions

    Article: Pan et al. Multiplexed Detection and Imaging of Intracellular mRNAs Using a Four Color Nanoprobe, Analytical Chemistry, 2013, 85, 10581-10588.

    Suggestions: read the article individually first (potentially multiple times) and make notes of items you do not understand, then re-read the article with your group.

    1. Look up and define any terms you were unfamiliar with, but are important for the comprehension of the paper. List the terms and definitions below.
















    1. Describe the relationship between concentration and signal for an experiment utilizing absorbance, fluorescence, and quenching. Be as specific as possible and draw a hypothetical plot of signal (include units in the axis label) vs. concentration.













    1. The authors chose to use four specific fluorophores, Alexa fluor 405, Alexa fluor 488, Cy3, and Cy5.
      1. Why did they choose the four they did? Do you see any potential problems and how would the results have differed if they had chosen the fluorophore DyLight 550 which has an excitation and emission wavelength of 562 nm and 576 nm, respectively. Support your answer with text from the paper.

      2. Using data presented in the paper rank these fluorophores in order of highest (1) to lowest (4) quantum yields. Explain your reasoning and be sure to define quantum yield in your explanation.

      3. Considering Figure S3. Estimate k’ for each fluorophore (include units). Which fluorophore would result in the highest sensitivity when used as a label? Show your work and explain your answer.

      4. Why do the fluorophores measure the emission intensity at a longer wavelength than the emission wavelengths (draw a figure if helpful)?

      5. Which fluorophore that the authors used required the most energy to excite? How much energy did a photon used to excite it possess?

    2. Under the Nuclease Assay section (top right of 10583) the last sentences states “fluorescence was measured at appropriate excitation wavelengths after the solution was cooled to room temperature (from 37 oC)”. Why did the authors cool the solution? (i.e. How do you think the fluorescent signal would have changed if the readings were taken immediately at 37 oC?) Explain your reasoning.




    1. The authors use a MTT assay to assess the cell viability and toxicity of the nanoparticles. Briefly describe a MTT assay and how it works to determine toxicity. A resazurin assay is another method to assess cell viability. Look up and briefly describe this assay as well. Compare and contrast the two assays.









    1. The authors use RT-PCR to confirm the nanoprobe results. Describe briefly how RT-PCR works and interpret the results presented in Figure S6, S7, and S9.













    In-Class Questions- Day 2

    Article: Pan et al. Multiplexed Detection and Imaging of Intracellular mRNAs Using a Four Color Nanoprobe, Analytical Chemistry, 2013, 85, 10581-10588.

    Discuss these questions with your group. You have roughly 5 minutes for discussion of the question and we will discuss as a class your responses.

    1. What is the overall goal of the sensor described in the paper and how do the authors achieve this analysis? Be sure to include any specifics that make this analysis possible.






    1. Referring to Figure 2 (pg 10584).
      1. Do you feel the MBs could be used in the quantification of each of the target analytes (all 4) and why or why not. If so draw vertical lines on the Figure 2 on the last page of the exam representing the high and low end of the dynamic range exhibited by each MB (use N/A if you feel there is no dynamic range).

      2. Which MB/mRNA pair shows the largest dynamic range and which is the smallest? You can assume each successive data point corresponds to a similar concentration across all of the plots.

      3. Do you expect the values (concentration and fluorescence) in Figure 2 and Figure s3 should directly correspond to one another? Explain your answer.




    1. Also referring to Figure 2.
      1. What is limiting the dynamic range of each MB and describe a method that could increase the dynamic range?

      2. How would you address a situation in which the target analyte’s concentration was over 200 nM?

    2. In the abstract and through the text the authors claim simultaneous detection of the 4 analytes. Define what simultaneous means to you. Is this detection truly simultaneous? If it is, explain. If it is not, what would need to happen to make it truly simultaneous and is this feasible?





    1. Describe the recognition element for the biosensor and briefly discuss the selectivity of each MB. Use data from the paper to support your answer.







    1. Do you feel the authors could increase the multiplexing capabilities? What would limit the number of analytes able to be detected and quantified by a single nanoprobe?








    Extra Practice

    1. You are now working to quantify the mRNA expression in two different cell types using the nanoprobe. Although the fluorophores are relatively well spaced in terms of the excitation and emission profiles there is small amount of overlap between the Cy3 and Cy5 emission profiles. You decide perform a calibration plot at each respective wavelength so that you can quantify both c-myc (Cy3) and GalNac-T (Cy5) mRNA simultaneously using the same nanoprobe. The results of your calibration plots are below.
        k’560(μM-1) k’688(μM-1)








      After lysing the cells and isolating the mRNA using a Qiagen mRNA isolation kit you then remove 10 μL of your concentrated mRNA and add it to 90 μL of the detection solution containing your nanoprobe. You measure the fluorescence and your solution has a fluorescence intensity of 46.3 RLU at 560 nm and 27.5 RLU at 688 nm.

      1. Why was the k’ determined at two different wavelengths for each fluorophore?


      2. What is the nM concentration of each mRNA?





















    1. The same instrument could have been used to collect the data under the Quantitation of Each MB Loaded on the Nanoprobe and MTT Assay subsections (pg 10583) by changing a few of the settings and specifics of the measurement. Describe and draw a diagram of an instrument that could be used for both measurements and how you would switch between the different fluorophores AND the two different experiments. Be as specific as possible (i.e. if you include a source what type of source).






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