# A progesterone biosensor derived from microbial screening

22 Nov.,2022

Progesterone Powder

### Materials

All DNA oligonucleotides were purchased from IDT Technologies. Progesterone (PRG), cholesterol (CHL), cortisol (CRT), aldosterone (ALD), estrone (ESN), 5β-pregnane-3ɑ,20-ɑ-diol (PRE), 5β-pregnane-3ɑ,20-ɑ-diol-glucuronide (PRE-Glu), and lysozyme were bought from Sigma Aldrich. Artificial urine DIN EN1616:199 was bought from Pickering Laboratories. RNAprotect Bacteria Reagent was bought from Qiagen. Proteinase K was bought from Roche.

Cadmium oxide (CdO; 99.95%, Alfa Aesar), sulfur (99.95%, ACROS Organics), 1-octadecene (ODE; 90% ACROS Organics), and oleylamine (80–90%) were bought from Fisher Scientific and used as purchased. Zinc acetate (Zn(Ac)2, 99.99%), selenium pellets (Se, 99.99%), trioctylphosphine (TOP, 97%), trioctylphosphine oxide (TOPO, 99%), oleic acid (OA, 90%), poly(isobutylene-alt-maleic anhydride)–6000 g mol−1 (PIMA), (2-aminoethyl)trimethylammonium chloride, histamine, triethylamine, and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) were obtained from Sigma-Aldrich. HPLC-grade solvents including hexanes (Fisher Scientific), methanol (Honeywell), anhydrous dimethyl sulfoxide (Sigma Aldrich) and chloroform (J.T. Baker) were bought and used without further purification. HEPES 1× is a solution of 25 mM of HEPES and 150 mM of NaCl, adjusted to pH 7.6.

### Strain selection

Pimelobacter simplex strain 6946 was purchased from ATCC and referenced with a corresponding GenBank accession number (CP009896.1). The strain is an obligate aerobe and was grown in media and conditions as recommended by ATCC.

### Strain characterization

To determine the doubling time of the strain, growth curves were generated (Supplementary Fig. 18). All growth curves were done in 100 µL per well volumes in 96 well flat clear bottom black polystyrene TC-treated microplates which were individually wrapped with a lid and sterile (Corning). Measurements were done by an Infinite M200 Pro (TECAN) spectrophotometer at the temperature suited for P. simplex. Readings were performed over 96 cycles of 15 min each at 600 nm absorbance with 25 flashes in a 3 × 3 (XY-Line) type reads per well. In between reads there was orbital shaking at 150 rpm frequency for a total of 10 min. To first characterize the growth alone, a ½ serial dilution of 9 concentrations from 0.5–0.0020 OD600 nm were prepared in the respective media. Then each concentration was measured as previously described by the TECAN microplate reader and normalized against a media background control in technical triplicate.

### Solvent exposure

Once an appropriate starting cell concentration was chosen, a secondary growth curve was performed to test the toxicity levels of the solvents used to dissolve steroids of interest (Supplementary Fig. 18). Since the steroids used are heavily hydrophobic, they often need to be dissolved in organic solvents such as DMSO or ethanol which are toxic to bacteria at high concentrations. P. simplex was incubated under microplate reader conditions previously mentioned at a starting OD determined by the first growth curve with DMSO, ethanol, or H20 at ½ serial dilutions for a total of eight concentrations (50–0.39%) tested in technical triplicate. Two controls were included per solvent; a positive control without solvent and a media control which allowed for appropriate normalization. Solvent exposure growth curves allowed for choosing the maximum amount of solvent concentration that strains would sustain while maintaining relative viability in order to determine a range of steroid concentrations which could be used.

### Steroid exposure

A tertiary growth curve was performed to test the toxicity levels of steroid specific to P. simplex (Supplementary Fig. 18). The strain was incubated under microplate reader conditions previously mentioned at a starting OD determined by the first growth curve and the highest solvent concentration corresponding to the steroid of interest with steroid at ½ serial dilutions for a total of seven concentrations tested in singlet. Testosterone, progesterone, 17β-estradiol, hydrocortisone, and aldosterone were all dissolved in ethanol while estrone was dissolved in DMSO. Three controls were included for each steroid; a positive control with the highest tolerable solvent concentration (%), a positive control with media, and a media control which allowed for normalization. Steroid exposure growth curves allowed for choosing the maximum amount of steroid concentration that P. simplex would sustain while maintaining relative viability.

### RNA extraction

Cells were grown in 6 mL volumes of media at the OD, solvent, and steroid concentrations found from the growth curves in 14 mL polypropylene round-bottom tubes. The same controls as in the steroid exposure growth curve were used for setting up RNA extraction samples. The cells were incubated at their corresponding temperature with continuous orbital shaking at 150 rpm until mid-log phase and the beginning of stationary phase from the start of inoculation. Afterward, samples were removed and a 1:1 ratio of RNAprotect Bacteria Reagent was added followed by spinning down at 4 °C for 10 min at 4000 × g. Supernatant was removed and the pellet re-suspended in 300 µL of RNAprotect and transferred into 2.0 mL Safe-Lock Tubes. The samples were then spun down once again at 4 °C for 10 min at 10,000 × g. Once the supernatant has been removed the samples were placed on ice and ready for RNA extraction. RNA extraction was done by Qiacube set to the RNeasy Protect Bacteria Mini Kit protocol of bacterial cell pellet with enzymatic lysis. Tube A was prepared as described except with the addition of 150 mg mL−1 lysozyme and 20 mg mL−1 proteinase K all diluted in 1× TE buffer. RNA samples were subsequently quantified using Qubit RNA HS Assay Kit (Thermo Fisher Scientific) and analyzed using a RNA 6000 Pico Kit (Agilent) in a 2100 Bioanalyzer (Agilent). RNA samples were either immediately used for RNA-Seq library preparation or stored long-term at −80 °C.

### RNA-Seq library preparation

After RNA samples have been quantified and analyzed they were DNase treated using a TURBO DNase 2 U µL−1 (Thermo Fisher Scientific) and cleaned using Agencourt RNAClean XP SPRI beads (Beckman Coulter). RNA-Seq libraries were then produced from these samples using a slightly modified ScriptSeq v2 RNA-Seq Library Preparation Kit (Illumina) ensuring use of unique index primers through ScriptSeq Index PCR Primers (Sets 1–4) 48 rxns/set (Illumina). Libraries were quantified by both a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and by High Sensitivity DNA Kit (Agilent). The resulting molarity was then used to determine at what concentration samples should be pooled to (either 1, 2, or 4 nM). After a desired pooling concentration was chosen, the samples were diluted to that particular molarity and 2 µL of each sample dilution was added into a single tube and submitted to the Boston University Microarray and Sequencing Resource Core Facility. Whole transcriptome RNA sequencing was performed by a NextSeq 500 (Illumina) at high output (400 M reads) with 75 bp paired end sequencing read length. The data was then analyzed in-house through a proprietary Galagan Lab pipeline.

### RNA-Seq data analysis

Adapter sequences were removed from reads and low quality bases trimmed from both ends using Cutadapt48. Reads were aligned to the reference genome with Bowtie249. BAM files were sorted and indexed using Samtools50. Transcript assembly and expression quantification was performed using Cufflinks51. All resulting raw expression counts were normalized as a group using deseq52. A custom matlab script was then used to calculate fold changes of normalized counts for each gene between each sterol exposure experiment and its corresponding vehicle control.

### Protein expression and purification

SRTF1 coding sequence codon-optimized for expression in Escherichia coli was ordered from Integrated DNA technologies with appropriate up-stream and down-stream fusion sequences for cloning into the pRham C-His Kan vector (Lucigen), resuspended in 1× TE buffer, and 45 ng heat shock transformed into chemically competent E. cloni 10 G (Lucigen) along with 25 ng of linearized pRham C-His Kan Vector (Lucigen). Cells were grown on LB agar + 50 µg mL−1 kanamycin overnight at 37 °C. Resulting colonies were grown in 5 mL LB + kanamycin, plasmid purified using Qiagen Miniprep Kit, and CDS insertion verified through Sanger Sequencing using forward primer rRham F and reverse primer pEtite R (Lucigen).

Protein was expressed in E. cloni 10 G cells grown overnight from a singly colony. Five milliliters overnight LB + kanamycin culture was diluted in 250 mL of fresh LB + kanamycin, then allowed to grow to an OD600 of approximately 0.6 shaking at 37 °C. Two and a half milliliters of filter sterilized 20% w/v rhamnose was then added to induce protein expression, and culture was shaken for 4 h at 37 °C. Culture was removed then removed from the incubator and stored at 4 °C overnight. Culture was then pelleted by centrifugation and frozen overnight.

Protein was purified using the Ni-NTA Fast Start kit (Qiagen). Cells were lysed using 10 mL of the provided Native Lysis Buffer supplemented with 10 mg lysozyme and 250 U benzonase as specified in the manufacturer’s protocol. Pellet was resuspended by stirring and pipetting up and down with a 25 mL graduated pipette. Cells were lysed on ice for 1 h, with gentle swirling every 20 min. Some preparations were highly viscous after the 1 h incubation, likely due to incomplete nuclease activity. Viscous lysates had an additional 250 U benzonase added to them and were incubated for twenty more minutes. All samples treated with additional benzonase had their viscosity reduced to normal after this additional incubation. Lysate was centrifuged at 14,000 × g for 30 min at 4 °C. Lysate supernatant was applied to a pre-drained Ni-NTA column and allowed to run through at room temperature. Column was washed three times with 4 mL of 3 mL of Native Wash Buffer (NEB), then eluted twice with 1 mL each of Elution Buffer (NEB).

Protein was present in both elution fractions off the column, so elutions 1 and 2 were pooled for desalting. Protein was either dialyzed against the destination buffer using 0.5–3 mL 3500 MWCO Slide-a-Lyzer (ThermoFisher), twice for 2 h at room temperature with magnetic stirring, then overnight at 4 °C without stirring, or using Amicon Ultra-0.5 centrifugal filters with a 10 kDa MW cutoff. Elutions were added to the column 0.5 mL at a time, spun at 14,000 × g at 4 °C until only approximately 75 µL remained, around 30 min. Destination buffer was added to the 0.5 mL mark, and centrifugation repeated. This process was repeated 3 additional times, for a total of 4 times.

Destination buffer for protein used in BLI, iv-ChIP-seq, and QD-TF-FRET assays was Tris-buffered saline pH 7.4. Protein concentration was quantified using a Qubit Protein Assay kit (ThermoFisher), then aliquoted and frozen at −80 °C until used.

### In vitro ChIP-Seq

We developed an in vitro ChIP-Seq assay similar to previously published in-vitro DNA precipitation protocosl53,54. P. simplex picked from a single colony on M3 Agar was grown in M3 Broth at room temperature with gentle shaking for 3 days until culture was turbid. One milliliter aliquots of culture were pelleted and genomic DNA was extracted using the GenElute Bacterial Genomic DNA kit (Sigma-Aldrich) using the included Gram-Positive Lysis solution and associated protocol adjustments for Gram positive bacteria. Purified gDNA was sheared into approximately 200 bp fragments in 130 μL aliquots using a Covaris S2 sonicator using a duty cycle of 20%, an intensity of 5, 200 cycles per burst, a time of 60 s, and 12 total cycles.

Purification of fragments of an appropriate size was done using AgenCourt AmpPure XP beads. 1.8× volume of bead slurry was added to the sonicated DNA and mixed by pipetting, followed by a five-minute incubation at room temperature. The DNA/slurry mixture was then placed on a magnetic rack for 10 min until all beads were pulled to the side of the tube. Supernatant was aspirated, and beads were washed twice with 200 μL 70% ethanol while still on the magnetic rack. Beads were air dried for 10 min, then resuspended in 100 μL of water, mixed by pipetting, and incubated at room temperature for 10 min. Beads were pulled down by placing on a magnetic rack for 10 min, then the supernatant and eluted DNA was pipetted off and saved. Fragment length was confirmed through 1% agarose gel electrophoresis in 1× TBE.

Fifty nanogram of sheared DNA was mixed with 500 ng purified SRTF1-CHis to a final volume of 500 μL in 1× IPP150 (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% NP40). Mock ChIP reactions were done by omitting the purified protein. Mixture was incubated at room temperature for 1 h, then 3 μL of 6×-His Tag Monoclonal Antibody His.H8 (Thermofisher Scientific) was added. Mixture was then incubated at 4 °C overnight with rocking.

Incubated mixture was then mixed with 5 μL of Protein G agarose beads (Pierce) that had been washed with 1 mL of IPP150 and pelleted by centrifugation for 2 min at 2000 × g. Bead mixture was incubated on a rocking platform for half an hour at 4 °C, then an hour and a half at room temperature. Beads were washed 3 times with 1 mL of IPP150, mixed by rocking each time. Wash buffer was removed by pelleting beads at 2000 × g for 2 min and pipetting off supernatant. DNA was eluted by adding 150 μL of “Elution From Beads” buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS) to pelleted beads and incubating at 65 °C for 15 min. Elution was repeated with a 5 min 65 °C incubation. Elutions were pooled, then purified using a QIAquick PCR Purification Kit (Qiagen), eluting with 30 μL of buffer EB.

Barcode adapters were ligated to immunoprecipitated DNA using the NEBNext Ultra II DNA Library Prep Kit for Illumina. As starting gDNA had been pre-sheared before immunoprecipitation, size selection was performed after 10 cycles of PCR barcoding. Library size and integrity was validated with an Agilent DNA 1000 Kit on an Agilent Bioanalyzer.

Library was sequenced on an Illumina NextSeq 500 to a depth of 4–10 M reads per sample. Illumina adapter sequences were removed with Cutadapt, and reads were aligned to the P. simplex VKM Ac-2033D genome CP009896.1 using Bowtie2.

### BioLayer interferometry

Single stranded DNA oligos containing SRTF1 binding sites or control sequences were ordered from IDT. Forward strands with 5′ biotin were annealed to reverse strands by mixing to a final concentration of 10 µM in Annealing Buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl), heating to 95 °C, then slowly letting them cool to room temperature.

All BLI steps were performed in 1× BLI Buffer (28 mM Tris-HCl pH 8.0, 5 mM MgCl, 4.25% Glycerol, 25 mM NaCl, 1.67 mg mL−1 BSA) at 30 °C in a ForteBio OctetRed96. For DNA association assays, SA tips were baselined in buffer, then dsDNA was loaded on by dipping into wells containing 75 nM DNA in buffer. DNA-coated tips were baselined again in buffer, then dipped in buffer containing varying concentrations of SRTF1 and allowed to reach equilibrium. Tips were then dipped in buffer for complex dissociation. Dissociation of DNA from the tip was controlled by subtracting the trace from a DNA-coated tip that was not exposed to SRTF1. Trace Y-Axes were aligned to the last 5 s of the second baseline step, and noise removed by Savitzsky-Golay filtering. Association and dissociation data were fit to a Mass Transport model.

Hormone-induced dissociation was assayed using BLI by dipping DNA-coated SA tips in buffer containing 150 nM SRTF1 and allowing the reaction to reach equilibrium. Complex was dipped in buffer containing varying amounts of hormone. Curve traces for each sensor were rescaled such that the binding level at equilibrium was equal to 1.

### Quantum dots synthesis

Core/shell/shell quantum dots were made using our previously reported procedure55 and is described briefly here. The precursors used for this synthesis included: 0.2 M Cd(OA)2, 0.2 M Zn(OA)2, 0.2 M sulfur in ODE, and 1 M TOP:Se. For the sulfur and selenium precursors, the appropriate amount of anion was weighed and dissolved into either ODE or TOP at the desired concentration with gentle heating. Once the solutions were fully dissolved, the precursors were heated under vacuum at 120 °C for at least 1 h before use to remove traces of water. For Cd(OA)2 and Zn(OA)2, CdO or Zn(Ac)2 were weighed and added to oleic acid at a 1:4 molar ratio. The solutions were heated under vacuum at 120 °C until fully dissolved and diluted to a final concentration of 0.2 M with ODE. All precursors were stored under argon at room temperature. Both Cd(OA)2 and Zn(OA)2 are waxy solids at room temperature and were therefore heated to 120 °C for use in the QD synthesis.

For nucleation of CdSe cores, we used an air-free hot injection method. In brief, 1 g of TOPO, 8 mL of ODE, and 1.9 mL of 0.2 M Cd(OA)2 were loaded into a 100 mL round bottom flask (rbf) and placed under vacuum at room temperature for 30 min. The flask was heated to 80 °C and degassed by backfilling with argon and switching back to vacuum 3× over the course of 1 h. Once the solution had been sufficiently degassed, the flask was placed under active argon flow and heated to 300 °C. In an argon-filled glovebox, we pre-mixed 4 mL of 1 M TOP:Se, 3 mL of oleylamine, and 1 mL of ODE for injection into the Cd solution at 300 °C. The reaction temperature was set to 270 °C. After 3 mins, the flask was taken off of the heating element and allowed to cool to room temperature. The CdSe cores were precipitated from solution under air-free conditions using ethanol and methanol and re-dispersed in hexanes.

### Polymer synthesis

The polymer capping the QDs (P1) was synthesized using a slightly modified version of a previously reported procedure56. In a typical experiment, 180 mg of PIMA (poly(isobutylene-alt-maleic anhydride), 6000 g mol−1, 0.03 mmol, 1 equivalent) was dissolved in 3 mL anhydrous dimethyl sulfoxide at 45 °C. In parallel, 116 mg (2-aminoethyl)trimethylammonium chloride (0.66 mmol, 22 equivalents), 73 mg histamine (0.66 mmol, 22 equivalents), and 193 µL triethylamine (1.39 mmol, 46 equivalents) were dissolved in 1.5 mL anhydrous dimethyl sulfoxide at 50 °C. After complete dissolution of both solutions, the solution containing the amines was added with a syringe to the PIMA solution. The reaction was kept overnight at 45 °C. The polymer was purified by several precipitations in ethyl acetate. A white powder was obtained with 67% yield. 1 H NMR (500 MHz, D2O): δ(ppm) = 8.33 (s, 0.35 H, imidazole), 7.14 (s, 0.52 H, imidazole), 3.61 (0.63 H), 3.37 (2.24 H), 3.08 (s, 5.14 H, quaternary amine), 2.82 (1.13 H), 2.49 (1.26 H), 2.15 (1.01 H), 2–1 (m, 2.45 H), 0.86 (m, 6 H, 2CH3). FTIR shows the disappearance of the C=O stretch band of the anhydride at 1770 cm−1 and appearance of the C=O stretch of carboxylic acid and amide bond at 1710 cm−1 and1650 cm−1, respectively.

### Ligand exchange

QDs were transferred into water by capping their surface with the polymer P1. In a typical experiment, 475 µL QDs ([QD] = 3.0 µM, 1.4 nmol) were dissolved in 600 µL of chloroform. In parallel, 560 µL of P1 at 10 mg mL−1 in DMSO was diluted in 560 µL chloroform. The solution of P1 was added to the QD dispersion and the reaction proceeded overnight with vigorous stirring. The next day, 0.5 mL of 0.1 M NaOH was added and the dispersion quickly shaken by hand. The QDs nicely transferred to the upper water phase. The water phase was extracted and centrifuged at 3800 × g for 1 min. Then the supernatant was filtered through 100 nm PVDF and washed 3 times with 0.1 M NaHCO3 with a 100k ultra-centrifugal filter. QDs were recovered in 0.1 M NaHCO3 at a concentration around 5 µM.

### Sensor assembly

For a typical experiment, using a molar ratio of QD/TF/DNA = 1/4/18: 275 µL QDs at 0.15 µM in 1× HEPES with 1% BSA, were mixed with 275 µL SRTF1-his6 at 0.6 µM in 1× HEPES, at room temperature for 45 min. Double-stranded DNA labeled with a Cy5 fluorescent probe at the 3′ and 5′ ends (275 µL, 2.7 µM in 1× HEPES,) was added to the mixture. After 30 min, 220 µL of 1× HEPES, and 330 µL of 5× binding buffer (25 mM MgCl2, 25% glycerol, and 250 mg L−1 Invitrogen™ UltraPure™ Salmon Sperm DNA in 0.1 M Tris-HCl) were added and the mixture incubated for 15 min at RT.

Fluorescence measurements were recorded on a Horiba Nanolog spectrofluorometer equipped with a plate reader. Absorption spectra were recorded using a Nanodrop 2000c. Fifty microliter of the sensor (QD/TF/DNA) was split in 3 × 9 centrifuge tubes to which 10 µL of progesterone at the desired concentration is added. As such, the final concentration of QD/TF/DNA for the measurements is 25 nM/100 nM/450 nM. A 384-well plate was filled with 60 µL of each solution. The fluorescence intensity was monitored on a spectrofluorimeter from 535 nm to 800 nm with excitation at 400 nm and a 450 nm long-pass filter before the emission detector. Fluorescence intensity was spectrally deconvolved into FRET donor and acceptor components and total intensity used to calculate normalized FA/FD. Ratiometric analysis using single wavelength point measurements of FA and FD displayed equivalent accuracy to full deconvolution.

### Stability assays

For the stability assays, QDs, TF, and DNA were mixed together and stored in the conditions described (e.g., RT, at 4 °C, or in the freezer following lyophilization); in each case the sensors were protected from light to prevent photobleaching. HEPES 1× and 5× binding buffers were added to the sensor components before starting the progesterone titration. Lyophilized sensors were first recovered in ultra-pure water (same volume as sublimated during lyophilization process), then HEPES 1× and 5× binding buffer were added before the progesterone titration.

### Cross reactivity calculation

Cross-reactivity is calculated with the following equation57:

$${\mathrm{\% Cross}}\,{\mathrm{reactivity}}\,=\,\frac{{{\mathrm{IC}}_{50}{\mathrm{of}}\,{\mathrm{analyte}}}}{{{\mathrm{IC}}_{50}{\mathrm{of}}\,{\mathrm{cross}}\,-\,{\mathrm{reactant}}}}$$

Cross-reactivity assays were performed using the sensor 3 configuration in HEPES 1× with SRTF1 and the mutant SRTF1_MUT1.

### Limit of detection calculation

The detection limit is the smallest concentration or absolute amount of analyte that has a signal significantly larger than the signal arising from a reagent blank. Mathematically, the limit of detection in the signal domain (LD) is given by:

$$L_{\mathrm{D}}\,=\,{\mathrm{mean}}_{{\mathrm{blank}}} - 3.3 \times {\upsigma}_{{\mathrm{test}}}$$

where meanblank is the mean signal for a reagent blank and σtest is the pool standard deviation for all test samples in the dilution series, calculated as58:

$${\upsigma}_{{\mathrm{test}}} = \sqrt {\frac{{\mathop {\sum}\nolimits_{i = 1}^m {\sigma _i^2} }}{m}}$$

where σi is the standard deviation in signal intensities for n replicates of the ith test concentration, with a total of m different test concentrations.

The limit of detection (LOD) was calculated using the parameters of the fit with the non-linear equation for y = LD:

$${\mathrm{LOD}}\,=\,{\mathrm{IC}}_{50}\,\times\,\root {p} \of {{\frac{{A_1 - A_2}}{{L_{\mathrm{D}} - A_2}} - 1}}$$

The 95% Confidence Interval was calculated using Origin Pro Software.

### Artificial urine assays

Artificial urine composition: pH 6.6 ± 0.1, urea 25.0 g L−1, sodium chloride 9.0 g L−1, disodium hydrogen orthophosphate anhydrous 2.5 g L−1, potassium dihydrogen orthophosphate 2.5 g L−1, ammonium chloride 3.0 g L−1, creatinine 2.0 g L−1, sodium sulfite hydrated 3.0 g L−1.

For the artificial urine assays, QDs, TF, and DNA were mixed together in 1× HEPES before artificial urine and artificial urine + PRG was added such that 50% of the final volume comprised artificial urine.

Artificial urine at 37 °C: the sensor was assembled at RT then artificial urine and artificial urine + PRG were added at 37 °C.

For tests in artificial urine following lyophilization, QDs, TF and DNA were assembled in 1% BSA for QDs and ultra-pure water (no salts) and lyophilized. The sensor was recovered in artificial urine (same volume as sublimated during lyophilization process). Then artificial urine + PRG was added to the sensor.

A UV-LED (LED405E, 10 mW, Thorlabs) was attached to an SM1-threaded LED mount (S1LEDM, Thorlabs) that was screwed into one of the ports of the cuvette holder. The LED was powered with 3.3 V using an Arduino Uno. Two phototransistors (Digi-Key, 751-1057-ND) were used to detect the emitted light, one for each channel. In order to detect the Cy5 emission (670 nm), a 665 nm LP filter (Chromatech, ET655lp) was placed in front of one of the phototransistors to filter the light emitted by the Cy5 dye. In order to detect the QD emission (605 nm), a 600 nm BP filter (Edmund Optics, P/N 84785, 600 nm, FWHM 50 nm) was placed in front of the second phototransistor to filter the light emitted by the QDs.

Each one of the phototransistors was placed in a common-emitter phototransistor circuit (Supplementary Fig. 16). A DC power supply was used to power the circuit (Bk Precision, 1760A). During an experiment, the voltage drop across the phototransistor was monitored with a multimeter (Agilent34410A) that was connected to LabView, which enabled real-time recording of measurements. The device was enclosed in a metal Faraday cage.

The LED was turned on and allowed to warm up for at least 20 min prior to the experiment. One hundred microliter of sensor with a given concentration of progesterone was pipetted into the cuvette. The cuvette was inserted into the cuvette holder such that the 10 mm path length was parallel to the filters. Upon inserting the cuvette, a timer was started. A black cover was placed over the cuvette and a lid placed on the Faraday cage. At t = 10, 30 and 50 s, a Labview program was started to begin data collection for a duration of 10 s each. Each 10 s long period of data collection is counted as one technical replicate.

The following equations were used to calculate FA/FD for the benchtop device:

$$\frac{{\mathrm{F}}_{{\mathrm{A,}} {c,i}}}{{\mathrm{F}}_{{\mathrm{D,}}{c,i}}}\,=\,R_{c,i}\,=\,\frac{{{\mathrm{Cy}}5_{{\mathrm{signal}},c,i}}}{{{\mathrm{QD}}_{{\mathrm{signal}},c,i}}}$$

where

$$\begin{array}{l}{\mathrm{QD}}_{{\mathrm{signal}},c,i} = \left( {5 - V_{out,cy5,i}} \right)\\ {\mathrm{Cy}}5_{{\mathrm{signal}},c,i} = \left( {5 - V_{out,QD,i}} \right)\end{array}$$

c = the concentration of progesterone in the sample i = the replicate number for a given concentration (i.e., 1 is the first technical replicate, etc.)

The standard deviation of each ratio is calculated using the following equation:

$$\sigma _{R_{c,i}}\,=\,R_{c,i}\sqrt {\left( {\frac{\sigma _{{\mathrm{QD}}_{{\mathrm{signal,}}{c}}}}{{{\mathrm{QD}}_{{\mathrm{signal}},{{c}},i}}}} \right)^2 + \left( {\frac{{\sigma _{{\mathrm{Cy}}5_{{\mathrm{signal}},c}}}}{{{\mathrm{Cy}}5_{{\mathrm{signal}},{{c}},i}}}} \right)^2}$$

In order to calculate the error bars associated with the average of the Ratio, the following equation was used:

$$\sigma _{Rc} = \frac{{\sqrt 3 }}{3}\sqrt {\left( {\sigma _{Rc,1}} \right)^2 + \left( {\sigma _{Rc,2}} \right)^2 + \left( {\sigma _{Rc,3}} \right)^2}$$

### Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.