Electronic detection of a target based on enzymatic cleavage of a reporter moiety
Apparatuses, systems, and methods are disclosed for target detection based on collateral cleavage of a reporter by an enzyme. A biologically gated transistor may include a channel and a reporter moiety immobilized to the channel. The state of the reporter moiety may affect one or more output signals from the biologically gated transistor when excitation conditions are applied to the biologically gated transistor and a sample fluid is applied in contact with the channel. A sample fluid may include an enzyme configured to activate in response to a target nucleic acid to cleave the reporter moiety. Excitation circuitry may apply the excitation conditions, and measurement circuitry may measure output signals from the biologically gated transistor. An analysis module may determine a parameter relating to presence of the target nucleic acid, based on the one or more measurements.
1. A system comprising:
a biologically gated transistor comprising:
a channel formed from a two-dimensional material disposed on a dielectric layer of a chip, the two-dimensional material selected from graphene or molybdenum disulfide, wherein an upper surface of the channel is configured to contact a sample fluid;
a conductive source and a conductive drain that are covered with a dielectric that electrically insulates the conductive source and the conductive drain from the sample fluid so that current between the conductive source and the conductive drain is through the channel; and
a reporter moiety immobilized to the channel, biologically gated transistor having a liquid gate voltage adjusted via a counter electrode and measured by a reference electrode, and the biologically gate transistor being configured such that one or more output signals of the biologically gated transistor are affected by a state of the reporter moiety at or near the channel surface in response to application of a sample fluid in contact with the channel, the sample fluid comprising an enzyme configured to
activate, in response to a target nucleic acid, to cleave the reporter moiety;
excitation circuitry configured to apply adjustments to the liquid gate voltage to the sample liquid via the counter electrode;
measurement circuitry configured to perform measurements of the one or more output signals from the biologically gated transistor that are modulated by the state of the reporter moiety; and
an analysis module configured to determine a parameter relating to presence of the target nucleic acid, based on the measurements.
2. The system of claim 1 , wherein the reporter moiety comprises polymeric strands with first ends linked to the channel and second ends that are not linked to the channel.
3. The system of claim 2 , wherein the second ends are linked to linkable molecules such that in response to the linkable molecules diffusing into the sample fluid in response to the enzyme cleaving the reporter moiety a decrease in an effective thickness of the reporter molecules in channel region and/or a decrease in capacitance of the reporter molecules in the channel region is detected.
4. The system of claim 1 , wherein the reporter moiety comprises polymeric strands with first ends linked to the channel and second ends linked to the channel, such that catalytic activity of target-activated enzymes collaterally cleaves the polymeric strands such that cleaved ends of the polymeric strands extend farther away from the channel than when the strands were linked at both ends.
5. The system of claim 1 , wherein the reporter moiety is indirectly immobilized to the channel as a backbone of a hydrogel immobilized to the channel.
6. The system of claim 1 , wherein the reporter moiety is a reporter nucleic acid and the enzyme is a nuclease enzyme, the reporter nucleic acid comprising one of RNA, DNA, and single-stranded DNA, selected for immobilization to the channel of the biologically gated transistor based on collateral cleavage of the reporter nucleic acid by the nuclease enzyme being activated in response to presence of the target nucleic acid in the sample fluid.
7. The system of claim 1 , further comprising the enzyme, wherein prior to application of the sample fluid to the biologically gated transistor, the enzyme is provided as a reagent for preparation of the sample fluid or pre-applied to the biologically gated transistor.
8. The system of claim 7 , wherein the enzyme is an RNA-guided Cas enzyme with guide RNA corresponding to a selected target nucleic acid.
9. The system of claim 7 , wherein the enzyme is selected to have site-specific binding to the target nucleic acid and non-specific collateral cleavage of the reporter moiety.
10. The system of claim 1 , comprising a plurality of biologically gated transistors with the reporter moiety immobilized to channels, wherein:
the plurality of biologically gated transistors comprises the biologically gated transistor;
the excitation circuitry is configured to apply excitation conditions to the plurality of biologically gated transistors;
the measurement circuitry is configured to perform measurements for the plurality of biologically gated transistors; and
the analysis module is configured to determine parameters relating to the plurality of biologically gated transistors.
11. The system of claim 10 , wherein the biologically gated transistors are disposed in a two-dimensional array of droplet locations on a substrate.
12. The system of claim 10 , wherein the biologically gated transistors are disposed in a linear array of locations on a substrate.
13. The system of claim 1 , wherein:
the measurement circuitry is configured to perform a plurality of time-dependent measurements of output signals affected by the state of the reporter moiety; and
the analysis module is configured to characterize a parameter relating to the state of the reporter moiety over time, based on the plurality of time-dependent measurements.
14. The system of claim 1 , wherein the set of one or more output signals comprise at least the following: a Dirac voltage, a channel current, a resistance, a capacitance, and/or an effective dielectric thickness of a layer formed at the surface of the channel in response to the state of the reporter moiety.
15. The system of claim 1 , wherein the parameter relating to presence of the target nucleic acid comprises one or more of the following: an indication of the presence of the target nucleic acid in the sample fluid, a concentration of the target nucleic acid in the sample fluid, to what extent the reporter moiety was cleaved, and/or a rate of cleavage of the reporter molecule.
16. The system of claim 1 , wherein:
the target nucleic acid comprises non-amplified single stranded DNA or non-amplified double stranded DNA;
the enzyme selected for activation by the target nucleic acid comprises a Cas12 enzyme; and
the reporter moiety cleaved by the activated Cas 12 enzyme comprises single stranded DNA.
17. The system of claim 1 , wherein:
the target nucleic acid comprises non-amplified RNA;
the enzyme selected for activation by the target nucleic acid comprises a Cas13 enzyme; and
the reporter moiety cleaved by the activated Cas 13 enzyme comprises RNA.
18. The system of claim 1 , wherein:
the target nucleic acid comprises non-amplified double stranded viral RNA;
the enzyme selected for activation by the target nucleic acid comprises a RNase L enzyme; and
the reporter moiety cleaved by the activated RNase L enzyme comprises single stranded RNA.
19. The system of claim 1 , wherein:
the target nucleic acid comprises a non-amplified cancer microRNA sequence
the enzyme selected for activation by the target nucleic acid comprises a Cas enzyme with a guide RNA complementary to the cancer microRNA sequence; and
the reporter moiety cleaved by the activated Cas enzyme comprises single stranded RNA.
20. The system of claim 19 , wherein:
first ends of the reporter moiety cleaved by the activated Cas enzyme are linked to the channel via molecular linkers comprising a carboxylic acid functional group and a pyrene base that anchors to the channel; and
second ends of the reporter moiety cleaved by the activated Cas enzyme are not linked to the channel.
21. The system of claim 11 , further wherein for different droplet locations in the two-dimensional array:
channels of the plurality of biologically gated transistors for the different droplet locations are functionalized with the same reporter moiety;
the enzyme in a sample liquid for analysis at the different droplet locations is an RNA-guided Cas enzyme with a guide RNA corresponding to a different target nucleic acids for the different droplet locations;
differences in the parameters determined for the plurality of biologically gated transistors in the different droplet locations are determined in response to differences in the state of the reporter moiety in the different droplet locations in response to activation of the enzyme at the different droplet locations by presence of different target nucleic acids corresponding the guide RNA for the different droplet locations.