Absorption spectra: To obtain absorption spectra(300 nm – 700 nm) of pH indicators, 200 µl of testing solutions(0.5 x phosphate buffered saline (PBS)) with pH values ranging from 1 to12 were mixed with 5 µl of the particular pH indicator in microtiterplates (96-well clear polystyrene; Greiner Bio-One, Frickenhausen, Germany) andanalyzed in a spectrophotometer (Spectrafluor Plus infinite M200 pro, TECAN).For each halochromic pH indicator, a specific wavelength with a maximalchange in absorption over the tested pH range was chosen (phenol red:558 nm, bromcresol purple: 588 nm, methyl red: 516 nm,bromthymol blue: 592 nm, cresol red: 572 nm, phenolphthalein:555 nm) and used for further testing. Comparison of pH indicators: To directly compare the suitability of the differentpH indicators under Pen reaction conditions, varying enzyme amounts(5 ng ? 12.
5 µU; 10 ng ? 25 µU,15 ng ? 37.5 µU; 20 ng ? 50 µU,25 ng ? 62.5 µU, all diluted in 40 µl of 0.5 xPBS) were mixed with 40 µl of 10 mM penG or 20 mM penG (finalconcentrations: 5 mM penG and 10 mM penG in 0.25 x PBS,respectively) and 3 µl pH indicator. The absorption of each mixture wasmeasured at the pH indicator-specific wavelength for 15 min. Theabsorption change (OD/min) of each approach was inferred from the slope of thelinear section.
All tests were performed in repeated independent experimentseach with triplicates for every penG concentration.Detection of different?-lactam antibiotics: To test the ability of penicillinase to detect different?-lactam antibiotics, six different antibiotic solutions (100 µl, between 0 mMand 50 mM) were mixed with 50 mU or 100 mU penicillinase and 3 µl bromcresolpurple. The absorption of each mixture was measured at 588 nm for15 min. Following antibiotics were testet: penicillin G, tricarcillin,carbenicillin, ampicillin, cloxacillin and cefotexim. For analysis antibioticswere dissolved in 0.
25x PBS. Adsorption of viral carriers andenzyme coupling:5 µg biotinylated TMVCys nanorods (TMVCys/Bio) werediluted in 100 µl binding buffer (10 mM SPP pH 7.8 and 137 mMNaCl) and incubated in microtiter plates (clear F-bottom 96-well polystyrenehigh binding MICROLON®; No.655061, GreinerBio-One, Frickenhausen, Germany) at 4 °Cfor 16 h to allow adsorption of viral adapters to the well surfaces, followedby three washing steps (RT, 5 min, 1 x PBS-T (PBS, 0.05 %Tween-20)).
In approaches designed to discriminate between specific(streptavidin-biotin mediated) and unspecific (mere adsorption) binding, non-specificadhesion of enzymes was reduced by blocking the wells each with 200 µl2 % (w/v) bovine serum albumin (BSA) in 1 x PBS for 1 h atRT, followed by three consecutive washing steps as before. If not otherwisestated, these pretreated cavities were incubated with 1 U SA-Pen or1 U Pen, respectively, (? 0.4 µg enzyme) diluted in 100 µl1 x PBS, for 3 h at RT.
After enzyme immobilization, wells werewashed thoroughly 5-6 times as before. Determination of catalyticactivities: Fordetermination of the catalytic activity in each approach, 100 µl substratemix (10 mM penG in 0.25 x PBS pH 8.0 and 3 µl bromcresolpurple) were applied. Absorption was measured for each well at 588 nm for10 min. The absorption was plotted vs.
time and the absorption change(OD/min) was derived from the slope of the linear section. All tests werecarried out repeatedly with triplicates for every approach.Reusability: After preparation of sensor wellsas described above, initial catalytic activities were determined, followed bythree consecutive washing steps and storage in 1x PBS at 4 °C.Remaining activities were measured for each approach every 2-3 days inrepeated experiments with triplicates of each sensor well layout. Initialactivities were set to 100 % and the percentages of remaining activitieswere calculated and plotted versus the testing period.Detection limit: For determining the detection limitof the TMV-assisted penicillin sensors, wells were prepared as describedbefore and treated with substrate solutions with different penG concentrationsranging from 0 mM to 50 mM (triplicates, with a few duplicates; pHadjusted to pH 8.
0 before use). The absorption was measured for each well at588 nm for 10 min and the absorbance change (OD/min) determined. Differencesoccurring among the the distinct analyte concentrations were subjected tostatistical evaluation by unpaired t-testing. A p value of less than 0.05 wasconsidered to indicate a significant difference between the absorbance changesreached with varying penG amounts (* p < 0.05; ** p < 0.01;*** p < 0.001).