Colorimetric to biotin-linked TMV rods enabled selective high-surface

Colorimetric
detection of penicillin was performed with enzme-based biosensors employing
TMV-derived nanoadapter scaffolds. Streptavidin conjugation of a commercial
penicillinase and its binding to biotin-linked TMV rods enabled selective
high-surface density immobilization.

 

The
colorimetric detection of penicillinase activity is based on acidometry employing
the hydrolysis of penicillin with differential halochromic color changes of pH
indicators (Figure 1C). To identify the most suitable pH indicator, six pH
indicator dyes were tested: phenol red, bromcresol purple, methyl red,
bromthymol blue, cresol red and phenolphthalein, which showed a clear color
change between pH 3 and pH 10 (the expected working range of the
penicillinase biosensor) (Figure S1). To quantify the proton generation, the absorption
spectra of each pH indicator between pH 1 and pH 12 were measured to
determine the wavelength of maximal change (Table 2, Figure S2). Phenolphthalein
did not show any relevant color change in the analyte/enzyme mixtures. Best changes
(OD/min) (Figure S3) and ?OD values (ODstart – ODend) (Table
2) were obtained by bromcresol purple (?OD = 0.76), followed by phenol
red (?OD = 0.36), cresol red, methyl red and bromthymol blue (?OD values
from 0.1 to 0.17). With bromcresol purple, low enzyme amounts (5 ng) in low
penicillin concentrations (5 mM penG) were detectable (Figure S3).

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The enzyme
penicillinase was further tested for its ability to detect different ?-lactam
antibiotics. A spectrum of six different antibiotics was tested: penicillin G
(benzylpenicillin), tricarcillin (carboxypenicillin), carbenicillin
(carboxypenicillin), ampicillin (aminopenicillin), cloxacillin and cefotexim
(cephalosporine) (Figure S5). Penicillin G and tricarcillin (semisynthetic) are
injectable antibiotics in human medizine, carbenicillin and ampicillin are semisynthetic
broad-spectrum antibiotic, cloxacillin is a ?-lactamase resistant antibiotic
solely used in veterinary medicine, cefotaxim belongs to third generation broad-spectrum
cephalosporins. As expected, penicillinase was not able to detect cloxacillin
and cefotaxime, due to the inhibited hydrolysis of the ?-lactam ring. However,
low absorbance changes in approaches with 100 mU enzyme allowed detecten of
moderate ampicillin concentrations (Figure S5, B). Carbenicillin and
tricarcillin were easily detectable, however lower absorbance changes were
obtained due to the semisynthetic modifications of the ?-lactam backbone. The
penicillinase, used in this study, is able to detect at least 3 different
?-lactam antibiotics; however, it is very likely that Pen is also able to
detect a much higher number of natural and semisynthetic penicillin-derivates.

 

 

Adsorptive
binding of biotinylated TMV to the sensor surfaces of microtiter plate wells was
followed by enzyme immobilization by streptavidin-biotin binding (Figure 2A). To
this aim, the most surface-accessible cysteine residues of the TMVCys particles
were modified with maleimide-PEG11-biotin linker molecules (TMVCys/Bio),
with a biotinylation efficiency of 95 % corresponding to ?2000 biotin
anchors per virus rod (Figure S4A). To allow a strong and specific coupling
of Pen, enzyme molecules were conjugated with streptavidin (SA)-complexes,
resulting in SA-Pen (conjugation effiency: > 90 % of the Pen
molecules (Figure S4B)).

The
penicillinase activity was not impaired by the streptavidin conjugation, since 60 µU
of both SA-conjugated and free penicillinase in solution yielded equal absorption
changes of ?3 OD/min in the reaction mixture (Figure 2A). On polystyrene
microtiter plates immobilized TMVCys/Bio sticks demonstrated the requirement
of streptavidin for successful coupling of penicillinase. Both biotin equipment
of the TMV adapters as well as streptavidin conjugation of the penicillinase
was crucial for immobilization of significant amounts of enzymes, resulting in detectable
substrate conversion rates (Figure 2A). The successful enzyme decoration
of TMVCys/Bio sticks was verified by TEM of freely suspended
particles prepared in parallel. Enzyme conjugation to TMV adapter sticks was evidenced
by an increased diameter of 30 – 33 nm and intensified staining for
the TMV adapter sticks (Figure 2B).

 

 

The
catalytic activities of Pen and SA-Pen (measured as OD/min) were analyzed after
immobilization on solid supports in direct comparison between setups based on
direct (adsorptive, without carrier) binding and setups equipped with
biotinylated TMV nano-carriers as adapters. Equal input amounts of SA-Pen or
Pen (each 1 U) were applied for binding into microtiter plate cavities as
depicted in Figure 3: TMVCys/Bio tubes (No. 1, 5)
(5 µg), TMVCys/Bio tubes (5 µg) followed by BSA treatment
to reduce unspecific enzyme binding (No. 2, 6), bare plate cavities (No. 3, 7)
or cavities pretreated with BSA (No. 4, 8; see also Figure 3C). Following
optional TMV adapter binding, optional BSA blocking, subsequent incubation with
enzyme or enzyme SA-conjugate, and removal of unbound SA-Pen or Pen
thereafter, absorption changes reflecting the enzyme activity in each approach were
determined by spectrophotometry. Highest absorption changes over time were
obtained in TMV-assisted layouts enabling SA-Pen bioaffinity coupling.
Cavities equipped with TMVCys/Bio (No. 1) reached a maximum change
of 13 × 10-2 OD/min (Figure 3A),
due to enzyme immobilization primarily through biotin-streptavidin interaction.
The use of TMVCys/Bio and BSA blocking (No. 2) achieved a
slightly lower absorption change of 11 x 10?2 OD/min,
indicating that in this layout, solely biotin-specific immobilization of SA?Pen
occurred (and thus, a minor additional amount of enzyme had adsorbed to the
non-blocked polystyrene surface in layout No. 1). The blocking efficiency
of BSA was confirmed in the cavities with BSA blocking preceding enzyme
incubation (No. 4, 8), which did not exhibit any enzyme activity
after washing. In layouts lacking either the viral adapter (No. 3, 7),
or streptavidin-conjugation of the enzyme (No. 5, 6), only lower
absorption changes of around 8-9 x 10-2 OD/min were
obtained, underlining the efficiency of the TMV-biotin-streptavidin enzyme trapping
method.

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