Disposable aptamer-sensor aided by magnetic nanoparticle enrichment for detection of salivary cortisol variations in obstructive sleep apnea patients

We report a disposable point-of-care sensing platform specific to salivary cortisol detection. The sensor is inkjet printed on a paper substrate with a metalloporphyrin based macrocyclic catalyst ink that can electrochemically reduce cortisol, captured by aptamer functionalized magnetic nanoparticles. The sensor consists of a thin magnet disc, aligned at the back of the electrode, in order to populate the magnetic nanoparticle bound cortisol at the sensing electrode area. Proof of concept studies were performed to detect salivary cortisol levels in human subjects with high and low risks for obstructive sleep apnea (OSA). High selectivity was observed to salivary cortisol against a background of closely related steroids.

energy (−15.15 kcal/mol) with cortisol. Binding energies were computed as the difference between the energy of the complex and the energy of each molecule.
Binding energies were computed as the difference between the energy of the complex and the energy of each molecule. Among all the M-PPs, Cu-PP was found to have the highest activity for cortisol. In the triplet ground state, Cu-PP had the strongest binding energy (−15.15 kcal/mol), compared to Ru (-14.12 kcal/mol) and Ni porphyrin (-13   Experimentally, cortisol in presence of Cu-PP, were observed to have a reduction peak (Epc) at -0.45 V which is attributed to the the reduction of cortisol ( Ipc of 1.8x10 -4 A). However, no reduction peaks we observed at Epc -0.45 V in the absence of Cu-PP (Fig.  S1c).  shows how subjects were to place a cotton pellet sublingual for 5 seconds or until the pellet was thoroughly moist. Subjects were then asked to place the moist cotton pellet in the respective labeled tube, and stored in a freezer until time of collection (visit #2). Subject participants marked the time that each salivary sample were taken along with any activities occurring during that time in a study diary for two sample collection days (Fig. S3). Study diary was used to record time of samples taken, and allowed us to remark on any deviations that may have occurred in salivary cortisol levels.  Please answer the questions below to help us assess for possible sleep apnea, a condition in which your breathing pauses or stops for periods of time while you sleep. Sleep apnea can increase your risk for many health conditions. It can also increase your risk for breathing problems after surgery.

Yes No
Have you ever been diagnosed with obstructive sleep apnea (OSA)?   Are you currently being treated for OSA?   Are you aware of a family history of OSA?   Are you aware of clenching or grinding your teeth at night?  

ESS: Epworth Sleepiness Scale
How likely are you to doze off or fall asleep in the following situations, in contrast to just feeling tired? 0 = I would never doze 2 = I have a moderate chance of dozing 1 = I have a slight chance of dozing 3 = I have a high chance of dozing

Situation
Chance of Dozing 1. Sitting and reading 2. Watching TV 3. Sitting inactive in a public place (e.g. a theatre or a meeting) 4. As a passenger in a car for an hour without a break 5. Lying down to rest in the afternoon when circumstances permit 6. Sitting and talking to someone 7. Sitting quietly in a lunch without alcohol 8. In a car while stopped for a few minutes in traffic STOP -BANG Yes No

Snore
Do you snore loudly? (Louder than talking or loud enough to be heard behind a closed door?  

Tired
Do you often feel tired, fatigued or sleepy during daytime?  

Obstruction
Has anyone observed you stop breathing during your sleep?  

Pressure
Do you have or are you being treated for high blood pressure?  

BMI
Is your body mass index greater than 28?  

Age
Are you 50 years old or older?  

Neck
Are you a male with a neck circumference greater than 17 inches, or a female with a neck circumference greater than 16 inches?  

Gender
Are you a male?  TGG TTAGCG TAT GTC ATT TAC GGACC-FAM-3'. The florescence intensity from the MNP-AptC-FAM was used to determine aptamer loading on nanoparticles. We observed a strong correlation between the amount of aptamers loaded on the nanoparticles and the initial concentration of S-MNp in the reagent buffer. Fluorescence micrographs (Fig  S4) reveal that the nanoparticles immobilized using 1 nM of aptamers loaded ~ 85 % more aptamers than the nanoparticles loaded using a 100 pM stock of aptamers.
Comparative studies also confirmed that the lower detection limit as well as the linear range was also a function of aptamer loading. Fluorescence microscopy images were taken using a Zeiss Axio Imager A1 fluorescence microscope with a Canon G6 digital camera attached. SEM and TEM images also confirmed that overloading nanoparticles with aptamers can lead to irreversible nanoparticle aggregation. Magnetic nanoparticles has been used successfully as solid phase support in immunoassays. Their ability to isolate various molecules in applications such as ribonucleic acid (RNA) purification, magnetic cell separation, magnetic particle enzyme immunoassay (EIA) and many other applications makes them beneficial to molecular and cellular isolations. The estimated oligonucleotide loading capacity of magnetic nanoparticles based on the diameter and concentration of nanoparticles is 7 x 10 12 aptamers/cm 2 for a stock solution of 1 nM AptC.

S5: Aptamer loading:
The quantitative determination of aptamer loading was obtained by measuring the initial aptamer concentration before and after immobilization by correcting for the dilution factor. Aptamer concentrations were determined from A260 as the peak at 260 nm corresponds to the DNA absorption. The absorbance of a solution increases as attenuation of the beam increases. Absorbance is directly proportional to the path length and the concentration of the absorbing species. we observed that AptC (1) which had an average aptamer density of 7.5 x 10 12 aptamers/cm 2 displayed a limit of detection(LOD) of 10 pM while AptC (10) which had 35% more aptamers than AptC (1) had a limit of detection of 30 pM (Fig. S5-d).