Proposed kinetic improvements to a Zika Biosensor developed by the Collins Lab at MIT that would reduce time of detection by 80% with a cost increase of only $0.01 per reaction.
1. German Soto, Katie Notarangelo, Anastasia Neuman
Kinetic Engineering Improvements to the Collins’ Lab ZIKA Biosensor
Although Zika rarely causes death in patients, it can be passed from a pregnant mother to
her fetus and cause microcephaly and other severe fetal brain defects. The full complications of
this virus are still unknown, particularly for fetuses. In addition to spread from pregnant mother
to fetus, Zika can be spread through sexual transmission and blood transfusion (Center for
Disease Control and Prevention, 2017). For these reasons, it is important to have a rapid and
inexpensive diagnosis tool for Zika to prevent outbreak originating from uninformed patients and
particularly to reduce spread of the disease to pregnant women. Zika viral loads have been
reported as high as 202 x 10^6 copies/mL (365 fM) in urine (Gourinat et al., 2015). However,
viral loads in saliva and serum are reportedly even lower, with 3 x 10^6 copies/mL (4.9 fM)
(Barzon et al., 2016) documented in patient saliva and 2.5 x 10^6 copies/mL (4.1 fM) (Zika
Experimental Science Team, 2016) and 7.2 x 10^5 copies/mL (1.2 fM) (Lanciotti et al., 2008) in
primate serum, respectively. Previous sensors displayed specificity for their respective Zika
RNA trigger, they were unable to detect clinically relevant RNA concentration. Consequently,
following the recent outbreak of Zika in the United States, the Collins lab developed a sensor to
detect Zika virus using programmable biomolecular components with a much higher specificity,
detecting Zika at concentrations as low as 3 fM (Pardee et al., 2016). The development method
used for this sensor can be translated to other viral diseases, providing a platform for rapid
response to any emerging outbreak.
In the Collin’s Lab Zika biosensor, the analyte being measured is viral Zika RNA. It is
sensed by a programmable RNA sensor called a toehold switch (Figure 1). When the target Zika
RNA binds a complementary region of RNA in the toehold switch, it frees a ribosome binding
sequence and start codon that promote the translation of the reporter gene, LacZ. LacZ mediates
a color change by converting chlorophenol red-beta-D-galactopyranoside (yellow) to
chlorophenol red (purple). The biosensor relies on a colorimetric output that can be read by the
2. naked eye or with a low-cost, battery-powered companion reader. There is an optional, portable
electronic reader that can be paired with the biosensor that provides quantitative measurements.
The electronic reader is manufactured using open-source code, laser-cut acrylic housing, and
readily-available consumer components, such as lithium-ion batteries. An overview of this
process is shown in Figure 2.
Figure 1. The toehold switch in this Zika-detecting device is
designed to complement the Zika trigger RNA. a) The switch RNA
within the toehold is complementary to the trigger RNA, which
here is Zika viral RNA. b) This shows the trigger RNA binding to
the toehold switch RNA. c) When the trigger RNA binds, a
ribosome binds to the ribosome binding site (RBS), and the
previously-repressed LacZ gene is able to be translated. (Image,
Green et al.)
3. Figure 2. The design of the biosensor begins with the development of the toehold switch
chemistry. The toehold is designed to specifically match the trigger sequence of the Zika
RNA, and the dissociation constant (k_D) of the trigger RNA and complementary toehold is
dependent on the length of the binding sequence, which is typically ~30 nucleotides.
Following sample collection, the Zika RNA is extracted and amplified via NASBA. The
paper-based sensor consists of freeze-dried toehold switches, that initiate a colorimetric
change.
The reaction occurring within this sensor that produces a measurable signal is the
conversion of Chlorophenol red-β-D-galactopyranoside to chlorophenol red, mediated by the
enzyme β-galactosidase. Na+
is used as a cofactor for this reaction. This enzyme is coded by the
reporter gene, LacZ, and is translated upon binding of Zika RNA to the toehold switch. The
balanced reaction for this conversion can be found below. The most important feature of this
reaction is the color change; Chlorophenol red-β-D-galactopyranoside has a yellow color while
chlorophenol red has a purple color, allowing the signal to be seen by the naked eye. The
Michaelis Constant (KM)of this β-galactosidase mediated reaction is about 990 umol/L, as
determined experimentally by Maceiczyk et al.
C25H22Cl2O10S (yellow color) + H2O -> C19H12Cl2O5S (purple color) + C6H12O6
4. The signal amplification process, Nucleic Acid Sequence-Based Amplification
(NASBA), is used to increase the sensitivity of the diagnostic platform. NASBA is an isothermal
RNA amplification technique, operating at 41° C, that starts with reverse transcription of the
target RNA (in our case the viral Zika RNA) that is mediated by a sequence-specific reverse
primer to create an RNA/DNA duplex. NASBA creates new target RNA that can be detected by
the toehold switch sensors by allowing allowing a T7 promoter to bind and initiate transcription
of the complementary strand, generating a double-stranded DNA product that serves as a
template for T7-mediated transcription and creation of copies of the target RNA sequence.
Specificity to the Zika genome was proven by testing the sensors with Dengue viral RNA
(51-59% similarity to Zika genome). No response to Dengue virus was seen. The system was
also proven to account for genetic variation among Zika RNA. The switches were shown to
tolerate up to 11% mismatch in nucleotide sequence, triggered successfully by both African and
Asian virus lineages. The possibility of false positive results is further minimized by
CRISPR-Cas9-mediated selection downstream of the amplification.
The researchers added a nucleic acid sequence-based amplification (NASBA) step in
order to increase the sensitivity of the system to meet this relevant range. With the added
NASBA reactions, the sensors reported detection of levels of Zika viral RNA as low as 3 fM.
Efforts were made to design a low-cost device with low-cost tests (Table 1). The process
takes a total of ~ 3.5 hours, with the bulk of the time going toward the NASBA amplification
(Table 2).
Table 1. Cost Estimates
Cost Estimates
NASBA Product $0.51/uL
Device $.10 - $1/test
5. Electronic Reader (Optional) $250
The cost of the device is derived from the 2mm paper disk, chlorophenol
red-β-D-galactopyranoside (Sigma-Aldrich), NEB solution A and B (NEB, PURExpress)
RNAse inhibitor (Roche, 03335402001; 0.5%), and linear DNA constructs encoding the toehold
sensors (.33 nM).
The NASBA Product cost is derived from human serum (Sigma H4522; 7%), Reaction
Buffer (Life Sciences NECB-24; 33.5%), Nucleotide Mix (Life Sciences NECN-24; 16.5%),
RNAse inhibitor (Roche, 03335402001; 0.5%), NASBA primer (2%), nuclease free water
(2.5%), RNA amplicon (20%), and Enzyme Mix (Life Sciences NEC-1-24; 25%).
Table 2. Time Estimates
Assay Time
Viral RNA Extraction 2 minutes
Amplification by NASBA 3 hours
Reactions and activation of signal 30 minutes
The device itself and RNA amplification process were engineered to accommodate
low-resource environments and transport time, as Zika and other viral infections are able to
spread rapidly in such areas without access to laboratory-grade equipment. The electronic reader
was made portable and powered by a rechargeable lithium ion battery. The shelf life of the
paper-based reactions and NASBA reactants were extended using a freeze-drying technique to
preserve the biomolecules during transport. The researchers also confirmed that the NASBA
process could successfully be made low-resource by substituting boiling for the initial heating
step.
It is known that there are several components within blood that are known to inhibit PCR
(Schrader et al., 2012), and similarly affect nucleic acid based diagnostics, including NABSA.
6. The inventors of the device overcome this challenge by diluting the sample (serum or plasma)
into water, which sufficiently removes the inhibitory effect in the diagnostic scheme. However,
this can be troublesome because the dilution step affects the overall sensitivity of the diagnostic
device. The inventors circumvent the detrimental effect of the dilution by increasing the NASBA
reaction time, shown to sufficiently compensate for the reduced sensitivity. An area of
improvement would be the elimination of this dilution step by using a different sample that
contains the virus at high enough concentrations for detection (i.e. saliva or urine) (Barzon et al.,
2016).
Further design improvements could be made via biomolecular, interfacial, or device
considerations. Sensitivity of the device could be increased by the biomolecular design of a
stronger LacZ promoter, which upon activation would produce more β-galactosidase, leading to
a stronger colorimetric signal from a lower analyte concentration. This could be addressed
thermodynamically by looking at the dissociation constant involved. The kinetics of the
β-galactosidase enzyme may be able to be improved.
Based on the Michaelis-Menten model of enzyme mechanics, the rate of production of
chlorophenol red, the reporter molecule, may be able to be increased by increasing the
concentration of substrate, the chlorophenol red-beta-D-galactopyranoside. Additionally, as
β-galactosidase is able to catalyze reactions of many different substrates, it may be possible to
find different reporting reactants that may have better kinetics, leading to shorter reaction times,
or reactants that may be more cost-effective.
One problem to address is the time scale required for the testing. While the timing of the
standardized NASBA procedure is the largest contributor to the length of the test, the reaction
itself has been reported to take over an hour. To determine whether the regime of this device is
diffusion- or kinetic-limited, we looked at the Damkohler number, Da:
7. Here, the reaction rate was defined as the rate constant multiplied by the concentration of
analyte, and the diffusion rate was calculated as the diffusivity of beta-galactosidase in water
multiplied by the interfacial area of the paper biosensor. Diffusivity was estimated using the
Stokes-Einstein equation, assuming a spherical protein (Edward):
Calculations gave a Damkohler number much less than 1, leading us to focus on the kinetics of
the biosensor reaction.
The reporter reaction is an enzyme-mediated colorimetric reaction, catalyzed by the
β-galactosidase enzyme, and can be modelled by the Michaelis-Menten kinetics:
This model provides a quantifiable relation between the rate of the enzymatic reaction
and adjustable parameters. Here, we hypothesized that increasing the concentration of the
chlorophenol red β-D-galactopyranoside substrate in the biosensor would increase the overall
rate of the reaction, therefore decreasing the amount of time required to get a response in use.
Maceiczyk et al. reports Michaelis-Menten constant values for this β-galactosidase
reaction as V_max = 2.18282 +/- 0.13769 and K_M = 0.90921 +/- 0.11661 (Figure _). From
8. these values and the 1.025 mM (0.6 mg/mL) concentration used by the Collins lab, we estimate
the reaction rate as 1.157 units, about 53% of the theoretical V_max. Using the
Michaelis-Menten design equation, we find that increasing the concentration by an order of
magnitude to 6.0 mg/mL, or 10.25 mM, 90% of the theoretical V_max can be achieved. This
would increase the reaction rate by about 80%, saving up to 48 minutes in testing time in the
device, while still reasonably below the solubility limit of chlorophenol red
beta-D-galactopyranoside in water (20 mg/mL).
Figure _. Plotted above is the relationship between reaction rate and concentration of
chlorophenol red beta-D galactopyranoside, based on results from Maceiczyk et al.
The current cost of the chlorophenol red-beta-D-galactopyranoside is $102.50/100 mg, or
$1.025/mg (Sigma-Aldrich). At the original concentration of .6 mg/ml, this would be a cost of
$.615/ml, or $.000615/ul. At the new suggested concentration of 6mg/ml, this would be a cost of
$6.15/ml, or $.00615/ul. The cell-free reactions containing chlorophenol
red-beta-D-galactopyranoside had a total volume of 1.8 ul. Although not all of the volume is this
substrate, we will conservatively estimate a volume of 2ul substrate used per reaction. This gives
an original cost of $.00123/reaction and a new cost of $.0123/reaction, or an increase of
$.01107/reaction. If we assume the lowest total test cost suggested in the paper, $.10/test, this
would be approximately a 10% increase in overall cost. If we assume the highest total test cost,
9. $1/test, this would only be approximately a 1% increase in total cost. This miniscule increase in
cost saves 48 minutes in testing time, making this a worthy investment.
In conclusion, we have improved the design of this biosensor by increasing the
concentration of the chlorophenol red-beta-D-galactopyranoside on the paper sensor by ten-fold,
from .6mg/ml to 6 mg/ml. This increases the rate of the toehold reaction and allows for visual
detection of the presence of Zika RNA in 12 minutes rather than one hour, an 80% decrease in
time. This time-save could allow hospitals to diagnose more patients in a smaller timeframe, and
ensure sick patients receive treatment faster.
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