BIOE658W-Biosensor Techniques Instrumentation and Applications

M. Rochelet, et al., Amperometric detection of extended-spectrum -lactamase activity: application to the characterization of resistant E. coli strains, Analyst, 140, 3551, 2015.

1. Answer the following questions about the experiments for the data represented by the open circles.

A. What is the biomarker that would be detected in the experiments represented?

Extended-spectrum β-lactamase (ESBL) 

B. What molecule is specifically providing the biorecognition in the experiments? 

Nitrocefin  

C. How is the biorecognition element immobilized onto the surface of the sensor system? 

By filtering through a membrane and placing it in a microwell 

D. What is the transduction mechanism; in other words, by what phenomenon is the presence of the target converted into a measured signal?  Be explicit but brief (no more than one sentence).

Catalytic hydrolysis of the β-lactam ring , shift in 

E. Excitation: what provides energy into the sensor to enable transduction?  Be as quantitative as possible.

Strong conjugation of both the β-lactam ring and the dihydro-thiazine ring with the dinitrostyrene group leads to a significant bathochromic shift of λmax

F. Measurement: how is the signal measured? What instrument is used?

Absorption spectroscopy 

Cyclic voltammetric measurements (CV and absorption spectrophotometry) to measure a colorimetric detection

G. True or false: the method has the potential to be used in real-time. ___F, it does measure the signal in real time, It required multiple step to do the measurement___________

Why or why not?

H. True or false: the method is label-free. ____F _________

I. True or false: the method is direct. _____F ________

2. Answer the following questions about the experiments represented in Fig. 2, specifically for the data represented by the open squares.

A. What is the transduction mechanism; in other words, by what phenomenon is the presence of the target converted into a measured signal?  Be explicit but brief (no more than one sentence).

Catalytic hydrolysis of the β-lactam ring 

Strong conjugation of both the β-lactam ring and the dihydro-thiazine ring with the dinitrostyrene group leads to a significant bathochromic shift of λmax

B. Excitation: what provides energy into the sensor to enable transduction? Be as quantitative as possible.  

Strong conjugation of both the β-lactam ring and the dihydro-thiazine ring with the dinitrostyrene group leads to a significant bathochromic shift of λmax

C. Measurement: how is the signal measured? What instrument is used.

Absorption spectroscopy 

Cyclic voltammetric measurements (CV and absorption spectrophotometry) to measure a colorimetric detection

G. True or false: the method has the potential to be used in real-time. _____F__ it does measure the signal in real time, It required multiple step to do the measurement___________

Why or why not?

H. True or false: the method is label-free. _____F_________

I. True or false: the method is direct. _____F________

3. Use the IUPAC approach to estimate the detection limit using the data in Figure 5.  Assume that the error bars on zero data point are the same as the 5 x 104 CFU/mL data point, assume that the error bars represent one standard deviation on each side of the mean, and assume that the error bar is 0.0075 A above and below the mean.  Show your work and state any additional assumptions.

Lowest concertation= ng/ml

LOD=3.3 x standard deviation of the regression line () /Slope(S)

S=(0.03-0.015)uA/2*10^5-0=0.75mA/CFU

LOD=3.3*0.0075uA/0.75mA/CFU~0.033 mCFU

4. Using the same assumptions as in #3, use the ISO definition and CLSI method to estimate the detection limit.  Show your work and state any additional assumptions.

5. Using the data in Fig. 5, determine the equilibrium dissociation constant Kd between the biomarker and biorecognition element (or, if you think this data is not sufficient to determine Kd, explain why it is not).

6. Recall the paper from Nie, et al. (Electrochemical sensing in paper-based microfluidic devices) that we read for this course in which the authors made an electrochemical sensor on paper for glucose detection.  Adapt that physical design to this ESBL sensor.  The sensor should be able to collect the sample by dipping it into the culture (or alternatively into a sample of lysed E. coli).  Assume that you found that the same immobilization works, and assume that you found that the same electrochemical mediator works.  Answer the following questions about this new design.  Also, feel free to make any sketches that will support your design.

A. What molecule is specifically providing the biorecognition in the experiments? 

B. How is the biorecognition element immobilized onto the surface of the sensor system? 

C. What is the transduction mechanism; in other words, by what phenomenon is the presence of the target converted into a measured signal?  Be explicit but brief (no more than one sentence).

D. Excitation: what provides energy into the sensor to enable transduction?  Be as quantitative as possible.

E. Measurement: how is the signal measured? What instrument is used?

Chronoamperometric & anodic stripping voltammetry measurement 

F. True or false: the method has the potential to be used in real-time. ______________ Why or why not?

G. True or false: the method is label-free. ______________

H. True or false: the method is direct. ______________

Refer to the following paper for questions 7-17:

L. Ge, et al., Three-dimensional paper-based electrochemiluminescence immunodevice for multiplexed measurement of biomarkers and point-of-care testing, Biomaterials, 33, 1024, 2012.

7. Answer the following questions about the experiments represented in Scheme 2.

A. What are the biomarkers that would be detected in the experiments represented?

Fluorescein isothiocyanate (FITC)-labeled CA125 

B. What molecules are specifically providing the biorecognition in the experiments? 

AFP & Fluorescein isothiocyanate (FITC)-labeled CA125 AFP, CA125, CA199, and CEA 

C. How is the biorecognition element immobilized onto the surface of the sensor system? 

Antibody on Chitosan

D. What is the transduction mechanism; in other words, by what phenomenon is the presence of the target converted into a measured signal?  Be explicit but brief (no more than one sentence).

ECL reaction 

E. Excitation: what provides energy into the sensor to enable transduction?  Be as quantitative as possible.  

ECL Peak and signal antibody was tagged with Ru(bpy), presence of antigen  

F. Measurement: how is the signal measured?  What instrument is used?

Measuring the intensity of ECL peak 

G. True or false: the method has the potential to be used in real-time ?  T,_ Measuring at real time  Why or why not?

H. True or false: the method is label-free. ______F________

I. True or false: the method is direct. ______T________

8. Use the IUPAC approach to estimate the detection limit using the data for CEA in Figure 4, but assume that the lowest-concentration data point is actually 0 ng/mL; for that point, use the error bars from the data points at the next higher concentration, which are more visible.  Assume that the error bars represent one standard deviation on each side of the mean, and assume that the error bar is 50 a.u. above and below the mean.  Show your work and state any additional assumptions.

Lowest concertation= 0ng/m

LOD=3.3 x standard deviation of the regression line () /Slope(S)

S= 500-700/20-50=400/300=1.33

LOD=3.3*0.5/1.3~1.2 

9. Using the same assumptions as in #8, use the ISO definition and CLSI method to estimate the detection limit.  Show your work and state any additional assumptions.

10.. Using the same assumptions again, determine the highest possible concentration that would result in a rate of 975 true negatives for every 1000 samples tested.  

11. Imagine that you had been developing the paper-based electrochemiluminescent (ECL) sensor for four cancer biomarkers (i.e., the same sensors discussed in this exam).  You then read the paper “Microfluidics-based diagnostics of infectious disease in the developing world.”  You decided to adapt this idea, but you continued to use the same transduction, excitation, measurement, and biorecognition method that you had been using for the paper-based ECL sensor.  To do so, you determined how to integrate electrodes into each of four sensing regions, and you also determined that passive adsorption worked for immobilization.  You used the sample loading procedure describe in the microfluidics paper.  

A. The sample is loaded in plugs.   Identify the necessary plugs below.  More plugs than necessary have been provided.  Try to minimize the number of reagents used for your method.  Assume that the biorecognition and surface blocking have already been performed.

B. Describe how excitation and measurement would work.  Include specific and quantitative details about signal levels and signal timing.

12. Imagine that you were evaluating this device for a CLIA waiver to use outside of a CLIA central lab.  The evaluation criteria for determining whether a diagnostic should receive a CLIA waiver (i.e., is sufficiently low in complexity) is given here: 

https://www.fda.gov/medical-devices/ivd-regulatory-assistance/clia-categorizations

Using this categorization, score the ECL version of the mChip for categories 2, 3, 4, and 7.  As directed on the CLIA website, give a score from 1 to 3 where 1 is low complexity and 3 is high complexity.  State the rationale for your score.  Complete credit is only granted for citing all of the correct reasons for the score.

A. Category 2 score____________

Rationale:

B. Category 3 score____________

Rationale:

C. Category 4 score____________

Rationale:

D. Category 7 score____________

Rationale:

13. Assume that the authors collected sufficient data for CEA to determine that the signal saturates at 4000 arbitrary units of ECL intensity.  Determine the equilibrium dissociation constant Kd between the immobilized biorecognition element and the biomarker (or, if you think this data is not sufficient to determine Kd, explain why it is not).

14. This paper and the MSD platform claim to provide multiplexed detection. But the approach is not quite the same. Comparing the work in this paper with an MSD assay using a Spector Imager, what are the differences in the excitation and measurement methods between the two, specifically related to multiplexed detection.

A. Excitation (if any differences)

B. Measurement (if any differences)

15. Does the MSD platform require a wash step after adding the labeling antibodies? Why or why not (credit only granted if the “why” is answered properly).

15. Does the system in this paper require a wash step after adding the labeling antibodies?

Why or why not (credit only granted if the “why” is answered properly).