Autonomous

Reovirus

Strain

Classification

Using

Filament-Coupled

Antibodies

We

previously

described

a

filament-based

antibody

recognition

assay

-LRB-

FARA

-RRB-

that

generates

ELISA-like

sandwich

structures

immobilized

on

a

filament

FARA

allows

the

coupling

of

antibodies

to

precise

locations

along

a

filament

on-line

fluorescence

detection

of

captured

pathogen

and

feedback-directed

filament

motion

These

properties

suggest

that

this

approach

might

be

useful

as

an

automated

means

to

rapidly

classify

unknown

pathogens

In

this

report

we

describe

validation

of

the

novel

decision

tree

aspect

of

this

technology

using

mammalian

reovirus

Based

on

available

antibodies

we

developed

a

decision

tree

algorithm

to

detect

virus

with

increasing

specificity

at

each

level

of

the

tree

Using

three

strains

of

reovirus

and

a

bacteriophage

control

our

system

correctly

classified

the

reovirus

strains

at

a

concentration

of

virions

ml

and

phage

at

virions

ml

Classification

of

reovirus

strain

type

Dearing

-LRB-

-RRB-

required

three

levels

of

testing

general

reovirus

classification

in

level

serotype

classification

in

level

and

final

strain

classification

in

level

Strain

also

required

three

levels

of

testing

before

a

final

classification

was

returned

in

level

Classification

of

strain

type

Lang

-LRB-

-RRB-

required

two

levels

of

testing

phage

detection

required

only

one

level

of

testing

for

classification

These

results

indicate

that

automated

pathogen

classification

using

FARA

is

feasible

Furthermore

the

simplicity

of

the

design

could

be

exploited

for

development

of

more

complex

sub-classification

networks

with

additional

levels

and

branches

Introduction

Identification

of

specific

pathogens

is

essential

for

the

selection

of

pathogen-specific

treatments

minimizing

the

spread

of

infection

and

monitoring

for

long-term

complications

Currently

specific

pathogen

identification

is

achieved

through

available

RT-PCR

and

antibody-based

strategies

In

the

clinic

these

tests

are

usually

applied

consecutively

to

evaluate

for

the

presence

of

the

most

likely

to

least

likely

pathogen

based

on

patient

findings

An

automated

classification

strategy

that

is

less

dependent

on

clinical

knowledge

but

achieves

rapid

accurate

identification

of

a

single

pathogen

from

among

a

group

of

possible

pathogens

is

currently

unavailable

Our

previously

described

filament-based

antibody

recognition

assay

-LRB-

FARA

-RRB-

employs

antibodies

immobilized

at

known

locations

along

a

filament

to

detect

specific

pathogens

The

filament

is

pulled

through

a

series

of

small

reaction

chambers

and

pathogens

if

present

are

captured

by

filament-bound

antibodies

Detection

of

pathogen

binding

is

achieved

by

using

a

fluorescently

labeled

second

antibody

specific

for

the

pathogen

FARA

was

first

reported

using

immobilized

antibody

to

detect

phage

This

virus

and

antibody

pair

provided

a

well-characterized

test

system

to

demonstrate

the

feasibility

of

a

filament-based

pathogen-detection

platform

However

in

this

first

generation

approach

the

filaments

were

removed

from

the

system

for

fluorescence

scanning

Subsequent

improvements

to

FARA

include

an

integrated

fluorescence

detector

and

a

feedback

algorithm

to

control

filament

position

The

integrated

detector

enables

adaptive

pathogen

detection

in

which

regions

of

interest

along

the

filament

can

be

reincubated

in

the

appropriate

reaction

chambers

to

increase

filament

fluorescence

when

the

initial

signal

is

low

In

this

report

we

describe

a

small-scale

test

of

the

use

of

a

simple

classification

tree

together

with

feedback-controlled

FARA

to

identify

four

viruses

The

feedback

feature

of

online

FARA

is

used

to

guide

the

selection

of

subsequent

tests

within

the

classification

tree

Unnecessary

tests

are

not

performed

and

each

subsequent

test

becomes

more

specific

for

a

single

pathogen

These

results

establish

FARA

as

a

robust

platform

for

classification

of

diverse

pathogens

Materials

and

Methods

Cells

and

Viruses

Murine

-LRB-

L

-RRB-

cells

were

cultured

in

suspension

in

Joklik

's

modified

Eagle

's

medium

supplemented

to

contain

fetal

bovine

serum

mM

l-glutamine

U

mL

penicillin

μg

mL

streptomycin

and

g

mL

amphotericin-B

Reovirus

strains

and

are

laboratory

stocks

is

a

monoreassortant

virus

isolated

from

L

cells

co-infected

with

and

contains

the

gene

segment

from

the

type

parental

strain

and

all

other

gene

segments

from

Reovirus

particles

were

purified

as

previously

described

L

cells

were

inoculated

with

second-passage

L-cell

lysate

stocks

of

twice

plaque-purified

reovirus

at

a

multiplicity

of

infection

of

plaque-forming

units

per

cell

Virus

was

purified

from

infected

cells

by

freon

extraction

and

CsCl-gradient

centrifugation

Purified

virus

was

obtained

from

the

Vanderbilt

Molecular

Recognition

Core

Antibodies

Mouse

monoclonal

antibodies

and

specific

for

reovirus

-LRB-

Table

-RRB-

were

purified

from

mouse

hybridoma

supernatants

using

Protein

A

column

chromatography

monoclonal

antibody

was

purchased

from

Amersham

Biosciences

-LRB-

Piscataway

NJ

-RRB-

Table

Antibody

specificity.AntibodyProtein

specificityReovirus

σ1

σ1

and

μ1

σ3

Antibodies

and

were

used

for

fluorescence

detection

of

virus

-LRB-

step

Table

-RRB-

and

were

labeled

with

Alexa

Fluor

or

Alexa

Fluor

-LRB-

or

Molecular

Probes

Eugene

OR

-RRB-

respectively

according

to

the

manufacturer

's

instructions

-LRB-

Molecular

Probes

-RRB-

Labeled

antibodies

were

purified

using

size-exclusion

chromatography

-LRB-

Amersham

Biosciences

-RRB-

Antibody

concentration

and

number

of

fluores

per

labeled

antibody

were

determined

by

using

absorbance

measurements

at

nm

and

the

peak

absorbance

wavelength

of

each

label

Aliquots

of

both

labeled

and

unlabeled

antibodies

were

stored

at

°C

and

working

solutions

were

kept

at

°C

Final

antibody

concentration

was

adjusted

immediately

prior

to

experiments

Table

The

five

reaction

chambers

in

FARA

processingChamberDescriptionSolutionIncubation

timeChamber

ID

Volume

-LRB-

mm

μl

-RRB-

IncubationUnknown

Antibody

IncubationFluorescently

Labeled

Detecting

-LRB-

-RRB-

was

added

to

PBS

as

a

blocking

agent

Filament

Preparation

Capture

antibodies

were

passively

adsorbed

to

the

filament

surface

in

groups

of

three

by

placing

the

filament

across

the

concave

teeth

of

a

PhastGel

sample

applicator

-LRB-

Amersham

Biosciences

-RRB-

-LRB-

Fig.

-RRB-

Three

capture-antibody

regions

corresponding

to

the

three

levels

of

testing

were

prepared

along

each

filament

using

three

applicator

combs

glued

end

to

end

Antibody

solution

pipetted

onto

the

filament

without

the

comb

spread

unimpeded

along

the

filament

Surface

tension

within

the

teeth

of

the

comb

overcame

this

tendency

and

produced

a

small

distinct

circumferential

band

of

immobilized

antibody

Antibody

was

spotted

in

a

volume

of

μL

and

allowed

to

passively

adsorb

to

the

filament

for

min

in

a

humidified

box

Following

incubation

at

°C

filaments

were

rinsed

in

phosphate

buffered

saline

with

Tween

-LRB-

PBS-T

-RRB-

and

threaded

through

the

reaction

chambers

for

virus

detection

experiments

Preliminary

experiments

were

performed

to

determine

the

optimal

concentration

of

each

capture

antibody

Figure

Schematic

of

a

device

for

adsorption

of

capture

antibodies

to

the

filament

The

filament

is

placed

within

the

concave

teeth

of

a

PhastGel

applicator

to

localize

the

spotted

antibody

to

a

small

circumferential

band

around

the

filament

Red

fingernail

polish

-LRB-

Poisonberry

Noxel

Corporartion

Hunt

Valley

MO

-RRB-

was

used

as

a

visible

and

fluorescent

fiducial

marker

to

identify

the

leading

and

trailing

edges

of

capture-antibody

regions

during

experiments

and

during

laser

scanning

The

polish

was

applied

by

pipette

between

the

teeth

of

the

comb

flanking

the

antibody

region

A

simple

bar

code

system

was

used

to

identify

each

of

the

three

capture-antibody

regions

The

first

and

second

regions

were

preceded

by

a

single

fiducial

marker

and

the

third

region

was

preceded

by

two

fiducial

markers

Micro-reaction

Chambers

Glass

microreaction

chambers

were

fashioned

from

inch

stock

tubing

into

mm

lengths

The

ends

were

flared

outward

to

facilitate

movement

of

the

filament

through

the

chambers

Interior

diameters

of

the

chambers

were

or

mm

depending

on

the

required

reaction

volume

-LRB-

Table

-RRB-

Chambers

were

carefully

positioned

in

a

straight

line

on

the

top

of

a

horizontal

aluminum

stage

using

machined

aluminum

mounts

Fine

adjustment

of

chamber

position

was

achieved

by

using

oblong

mounting

holes

on

each

mount

PBS-T

was

used

in

all

wash

chambers

and

for

virus

and

labeled

antibody

solutions

Reovirus

was

used

at

a

concentration

of

virions

mL

and

phage

was

used

at

a

concentration

of

virions

mL

Detecting

antibodies

were

present

in

the

detecting-antibody

chamber

at

a

concentration

of

approximately

μg

mL

for

each

antibody

in

the

three

antibody

mixture

Solutions

were

added

to

appropriate

chambers

at

the

initiation

of

experiments

and

used

for

all

three

levels

of

testing

-LRB-

Table

-RRB-

If

testing

proceeded

to

level

three

fluid

loss

from

the

chambers

was

monitored

and

replenished

if

necessary

Filament

Control

Movement

of

the

filament

and

therefore

the

antibody

bands

through

the

chambers

was

achieved

by

using

a

rotary

stage

to

wind

or

unwind

the

filament

around

a

spindle

A

small

weight

was

attached

to

the

opposite

end

of

the

filament

to

maintain

a

constant

tension

Filament

positioning

to

within

several

microns

was

achieved

by

using

a

rotary

stage

encoder

from

Yaskawa

Instruments

-LRB-

Waukegan

IL

-RRB-

and

a

custom

control

algorithm

written

as

a

LabView

Virtual

Instrument

-LRB-

VI

-RRB-

-LRB-

National

Instruments

Austin

TX

-RRB-

Parameters

including

filament

speed

and

residence

times

within

chambers

were

controlled

by

using

the

LabView

software

interface

Between

chambers

the

speed

of

the

filament

was

cm

sec

Within

each

chamber

the

capture-antibody

region

undergoing

processing

was

oscillated

cm

back

and

forth

at

a

speed

of

cm

sec

to

increase

interactions

between

immobilized

molecular

structures

attached

to

the

filament

and

the

molecular

species

in

solution

Within

the

detector

filament

speed

was

cm

sec

Lasers

and

PMTs

Filament

fluorescence

was

measured

by

passing

the

filament

between

two

diode

lasers

The

two

laser

excitation

sources

were

attached

to

either

side

of

a

detection

chamber

-LRB-

Fig.

-RRB-

Laser

-LRB-

nm

mW

diode

laser

Coherent

Santa

Clara

CA

-RRB-

was

used

to

excite

the

antibody

tag

Laser

-LRB-

nm

mW

diode-pumped

solid

state

laser

B&W

Tek

Inc.

Newark

DE

-RRB-

was

used

to

excite

the

antibody

tag

The

effective

power

of

laser

was

reduced

to

approximately

mW

by

using

a

polarizer

and

an

excitation

slit

Laser

was

not

attenuated

Two

Hamamatsu

photomultiplier

tubes

-LRB-

PMT

-RRB-

were

attached

to

the

top

and

bottom

of

the

sample

chamber

and

powered

by

and

V

signals

for

the

and

channels

respectively

Current

from

the

PMT

was

converted

to

voltage

by

using

transimpedance

amplifiers

that

amplified

the

signal

by

a

factor

of

for

and

for

Voltage

was

sampled

at

a

rate

of

samples

sec

by

using

a

digital

acquisition

board

from

National

Instruments

-LRB-

DaqPAD

-RRB-

Fluorescence

signal

was

acquired

as

a

function

of

filament

position

by

using

LabView

Following

fluorescence

scanning

for

most

experiments

filament

regions

were

cut

and

scanned

again

in

a

microarray

flatbed

scanner

-LRB-

GenePix

Axon

Instruments

Union

City

CA

-RRB-

Figure

Schematic

of

lasers

and

photomultipliers

-LRB-

PMT

-RRB-

to

detect

online

fluorescence

of

the

filament

The

filament

is

moved

-LRB-

arrow

-RRB-

through

a

rectangular

sample

chamber

with

a

laser

excitation

source

attached

to

either

side

Photomultiplier

tubes

are

attached

to

the

top

and

bottom

of

the

chamber

Filters

Filter

sets

were

placed

in

the

light

path

between

the

sample

chamber

and

the

photomultipliers

-LRB-

Fig.

-RRB-

Long-pass

filters

with

cutoffs

at

nm

-LRB-

Chroma

Rockingham

VT

-RRB-

and

nm

-LRB-

Melles

Griot

Rochester

NY

-RRB-

were

combined

to

reduce

reflected

laser

light

from

the

laser

For

the

channel

two

long-pass

filters

-LRB-

nm

cutoff

Melles

Griot

-RRB-

were

combined

with

a

bandpass

filter

centered

at

nm

-LRB-

nm

bandwidth

Chroma

-RRB-

to

reduce

reflected

light

Classification

Algorithm

The

LabView

program

coordinated

all

filament

movement

filament

scanning

and

feedback

control

Elements

of

the

feedback

control

are

described

by

the

nodes

of

the

decision

tree

used

to

classify

the

viruses

shown

in

Fig.

The

fluorescent

fiducial

markers

on

either

side

of

the

capture-antibody

region

produced

characteristically

sharp

emission

peaks

Since

the

physical

location

of

the

immobilized

antibody

positions

between

the

markers

was

known

experimental

conclusions

were

based

on

the

distance

of

the

first

detected

peak

from

the

initial

fluorescent

marker

Figure

algorithm

to

classify

virus

strains

The

filament

control

program

enters

the

decision

tree

from

the

top

at

level

and

based

on

the

type

of

virus

found

at

each

level

follows

different

branches

of

the

decision

tree

When

reovirus

is

detected

at

level

one

the

filament

is

moved

forward

to

the

next

antibody

region

for

level

testing

If

a

serotype

reovirus

strain

is

detected

-LRB-

or

-RRB-

at

level

testing

continues

to

level

to

distinguish

between

these

strains

The

filament-control

program

was

designed

to

find

each

peak

calculate

its

location

along

the

filament

and

make

a

decision

about

additional

tests

Spatially

localized

fluorescence

from

the

filament

was

measured

as

a

V

signal

from

the

transimpedance

amplifier

Voltage

data

from

the

scan

were

used

as

input

data

by

a

peak-detection

function

in

LabView

The

LabView

peak

detector

fit

a

quadratic

polynomial

to

sequential

sets

of

points

depending

on

a

width

parameter

entered

by

the

user

Data

were

then

compared

to

a

threshold

parameter

also

entered

by

the

user

to

identify

peaks

A

binary

decision

to

stop

or

continue

testing

was

based

on

the

location

and

number

of

peaks

found

Peak-detection

threshold

parameters

were

defined

as

V

-LRB-

approximately

three

times

background

-RRB-

with

a

width

of

data

points

which

corresponded

to

a

width

slightly

larger

than

the

physical

width

of

the

comb

tooth

A

common

set

of

parameters

was

used

in

all

experiments

based

on

previous

work

with

A

filament

region

containing

three

capture

antibodies

and

two

fiducial

markers

was

incubated

within

each

chamber

for

the

times

shown

in

Table

Each

of

the

capture-antibody

regions

contained

a

PBS

negative

control

in

the

first

position

In

level

one

the

second

position

was

a

mixture

of

and

-LRB-

mg

mL

mg

mL

-RRB-

The

third

position

was

-LRB-

mg

mL

-RRB-

In

levels

two

and

three

the

negative

control

position

was

followed

by

-LRB-

mg

mL

-RRB-

in

the

second

position

and

-LRB-

mg

mL

-RRB-

in

the

third

position

The

logic

encoded

in

the

decision

tree

shown

in

Fig.

was

followed

In

level

if

fluorescence

was

detected

from

the

position

of

the

first

region

of

the

filament

a

classification

of

virus

was

made

and

no

further

testing

was

conducted

If

fluorescence

was

detected

in

the

region

a

classification

of

reovirus

was

made

and

the

filament

was

advanced

to

the

appropriate

region

for

level

testing

where

a

more

specific

test

for

reovirus

was

performed

In

level

testing

fluorescence

from

the

region

indicated

a

serotype

reovirus

which

in

this

scheme

corresponded

to

reovirus

A

classification

of

represented

an

endpoint

in

the

decision

tree

and

testing

was

discontinued

Fluorescence

from

the

region

indicated

the

presence

of

a

serotype

reovirus

and

the

program

advanced

the

filament

for

further

subtyping

in

level

Level

testing

used

conjugated

antibody

as

the

detecting

antibody

and

a

second

detection

channel

with

a

green

excitation

laser

If

fluorescence

was

found

in

the

region

using

this

channel

a

classification

of

reovirus

was

made

and

testing

along

that

branch

ended

If

no

fluorescence

was

detected

from

the

second

channel

for

that

region

the

program

ended

with

a

classification

of

reovirus

If

no

peaks

were

found

between

the

fiducial

markers

a

message

was

generated

indicating

that

no

virus

was

found

Similarly

if

a

peak

was

detected

in

the

negative

control

region

of

the

filament

a

warning

message

was

generated

Although

theoretically

possible

this

situation

did

not

arise

during

testing

Results

FARA

utilizes

a

polyester

filament

with

circumferential

bands

of

immobilized

antibody

that

is

passed

through

a

series

of

five

glass

micro-reaction

chambers

that

are

similar

to

the

five

major

steps

of

an

ELISA

-LRB-

Table

-RRB-

In

the

first

reaction

chamber

of

the

FARA

approach

employed

in

this

study

capture

antibody

coupled

to

the

filament

was

rehydrated

In

this

chamber

the

filament

also

was

blocked

to

prevent

non-specific

binding

of

virus

In

the

second

chamber

the

immobilized

capture

antibody

was

incubated

with

virus

solution

In

this

chamber

if

virus

in

solution

bound

to

antibody

coupled

to

the

filament

then

the

virus

became

coupled

to

the

filament

via

this

interaction

In

the

third

chamber

non-specifically

bound

virus

was

removed

by

washing

before

incubation

with

a

fluorescently

labeled

detecting

antibody

in

the

fourth

chamber

A

final

wash

in

the

fifth

chamber

removed

non-specifically

bound

detecting

antibody

The

capture

antibody

region

was

then

passed

through

an

integrated

fluorescence

detector

In

these

experiments

the

test

virus

was

classified

with

greater

specificity

at

each

level

of

a

decision

tree

-LRB-

Fig.

-RRB-

Representative

fluorescence

signals

in

volts

obtained

during

testing

for

phage

and

reoviruses

and

are

shown

in

Fig.

Labels

for

the

PBS

control

position

and

the

antibodies

in

each

capture

antibody

position

are

shown

adjacent

to

the

filament

The

specificity

of

each

test

antibody

for

its

corresponding

virus

was

high

with

little

or

no

cross-reactivity

No

signal

was

detected

in

the

PBS

negative-control

position

Figure

Fluorescence

intensity

in

volts

as

a

function

of

filament

position

for

phage

-LRB-

A

-RRB-

and

reoviruses

-LRB-

B

-RRB-

-LRB-

C

-RRB-

and

-LRB-

D

-RRB-

The

immobilized

capture

antibody

positions

on

the

filament

are

labeled

within

each

panel

Captured

virus

was

detected

by

a

fluorescently

labeled

second

antibody

-LRB-

-LSB-

red

-RSB-

-LSB-

red

-RSB-

or

-LSB-

green

-RSB-

-RRB-

Corresponding

images

of

the

scanned

filaments

are

shown

at

the

top

of

each

graph

Each

panel

shows

a

representative

experiment

of

three

performed

Detection

of

was

achieved

by

level

testing

Strong

fluorescence

was

observed

in

the

position

but

not

in

the

or

PBS

positions

-LRB-

Fig.

-RRB-

This

finding

indicated

capture

of

the

phage

by

the

capture

antibody

For

detection

of

conjugated

was

used

Because

a

classification

of

was

made

the

other

two

capture

regions

of

the

filament

were

not

evaluated

Detection

of

the

reovirus

strains

employed

in

this

study

required

analysis

beyond

level

Level

testing

indicated

the

presence

of

a

reovirus

for

all

three

strains

with

a

peak

in

the

second

position

corresponding

to

virus

capture

by

the

antibody

mixture

In

level

testing

strain

was

detected

in

the

antibody

position

but

not

in

the

or

PBS

positions

-LRB-

Fig.

-RRB-

In

this

case

the

capture

and

detecting

antibodies

differed

Because

a

reovirus

classification

was

made

the

third

capture

region

of

the

filament

was

not

evaluated

For

reovirus

level

testing

indicated

the

presence

of

a

reovirus

with

a

peak

in

the

second

position

corresponding

to

virus

capture

by

-LRB-

data

not

shown

-RRB-

Level

testing

showed

a

distinct

peak

in

the

antibody

position

-LRB-

Fig.

red

curve

-RRB-

In

this

case

antibody

labeled

with

was

the

detecting

antibody

indicating

a

type

reovirus

Level

testing

showed

a

distinct

peak

in

the

antibody

position

-LRB-

Fig.

green

curve

-RRB-

In

this

case

antibody

labeled

with

was

the

detecting

antibody

Neither

curve

showed

cross

reactivity

of

reovirus

with

the

immobilized

region

or

the

negative-control

PBS

region

For

reovirus

level

testing

indicated

the

presence

of

a

reovirus

with

a

peak

in

the

second

position

corresponding

to

virus

capture

by

-LRB-

data

not

shown

-RRB-

Level

testing

yielded

a

distinct

peak

in

the

antibody

position

-LRB-

Fig.

red

curve

-RRB-

like

the

result

obtained

with

However

level

testing

with

did

not

yield

a

positive

signal

in

the

antibody

position

indicating

that

the

captured

virus

was

not

No

detectable

cross

reactivity

was

found

in

the

antibody

region

or

the

negative-control

PBS

region

Thus

the

virus

was

classified

as

Discussion

In

this

study

we

demonstrated

the

feasibility

of

a

FARA

pathogen-classification

approach

using

a

well-characterized

virus

system

Three

reovirus

strains

and

one

phage

were

successfully

classified

by

performing

sequential

antibody-binding

assays

directed

by

the

decision

tree

The

structure

of

the

decision

tree

was

based

on

published

characteristics

of

each

virus

strain

and

the

known

specificity

of

each

antibody

Because

the

capture

antibodies

were

arrayed

at

known

locations

along

the

filament

and

the

fluorescence

of

the

detection

antibodies

was

measured

as

a

function

of

filament

location

an

increase

in

filament

fluorescence

at

a

particular

location

indicated

antibody

interaction

with

the

virus

For

each

virus

strain

tested

the

expected

fluorescence

peaks

were

observed

and

the

correct

classification

was

made

Moreover

successful

transit

of

all

decision

tree

branches

was

demonstrated

The

biochemical

components

of

FARA

are

similar

to

those

used

in

standard

ELISAs

Both

assays

create

a

dual-antibody

sandwich

that

results

in

similar

detection

limits

for

for

FARA

and

ELISA

One

of

the

virus-specific

antibodies

acts

as

the

capture

antibody

and

the

second

acts

as

the

detecting

antibody

The

main

difference

in

the

biochemistry

of

the

two

assays

is

that

in

the

current

implementation

of

FARA

enzyme

amplification

is

not

utilized

Like

ELISA

FARA

utilizes

antibodies

adsorbed

to

the

surface

of

a

substrate

to

capture

virus

from

solution

The

capacity

of

bound

antibody

to

retain

its

antigen-binding

activity

is

essential

to

the

success

of

both

assays

Each

of

the

immobilized

test

antibodies

in

our

system

retained

functionality

and

bound

its

corresponding

virus

-LRB-

Fig.

-RRB-

The

absence

of

peaks

in

the

negative-control

positions

and

in

positions

occupied

by

antibodies

not

specific

to

the

test

virus

demonstrates

minimal

cross

reactivity

in

the

FARA

platform

employed

in

this

study

We

chose

reovirus

as

a

test

system

to

show

clinical

relevance

and

to

avoid

potential

safety

concerns

in

the

laboratory

Reovirus

has

been

used

as

a

model

to

study

mechanisms

of

viral

pathogenesis

in

mice

Although

it

is

a

human

pathogen

it

is

rarely

associated

with

human

disease

Many

reovirus

field-isolate

strains

have

been

characterized

and

a

broad

array

of

antibodies

are

available

that

recognize

reovirus

with

varying

specificity

We

found

that

not

every

antibody

tested

was

suitable

for

use

as

an

immobilized

capture

antibody

Neither

antibody

nor

bound

virus

when

immobilized

on

the

filament

even

after

virus

incubation

time

was

increased

to

greater

than

min

-LRB-

data

not

shown

-RRB-

It

is

possible

that

these

antibodies

undergo

conformational

changes

when

passively

adsorbed

to

a

solid

substrate

rendering

them

inactive

Alternatively

these

antibodies

may

be

incapable

of

antigen

binding

when

immobilized

as

a

consequence

of

steric

hindrance

Antibodies

and

recognize

the

μ1

and

σ3

proteins

respectively

which

form

the

bulk

of

the

viral

outer

capsid

It

is

possible

that

extension

of

the

viral

attachment

protein

σ1

may

shield

the

μ1

and

σ3

proteins

from

binding

to

the

and

antibodies

on

the

filament

surface

This

conclusion

also

is

consistent

with

our

observation

that

and

were

both

excellent

capture

antibodies

since

they

recognize

the

σ1

protein

which

extends

farthest

from

the

viral

capsid

The

failure

of

the

and

antibodies

to

function

as

effective

capture

antibodies

complicated

our

virus

detection

scheme

Although

is

not

serotype-specific

and

should

bind

all

reovirus

strains

we

could

not

use

this

antibody

as

the

immobilized

capture

antibody

for

level

testing

Antibodies

and

are

specific

for

serotype

and

serotype

reovirus

strains

respectively

therefore

we

immobilized

a

mixture

of

these

antibodies

for

detection

of

all

reovirus

isolates

in

level

Although

recognizes

most

type

strains

is

a

reassortant

virus

that

contains

a

gene

segment

from

As

a

result

does

not

recognize

and

is

specific

only

for

in

our

scheme

Since

could

not

be

used

as

a

capture

antibody

in

level

we

incorporated

a

fluorescently

labeled

antibody

as

a

detecting

antibody

in

solution

Although

the

nature

of

the

biochemical

interactions

is

similar

in

FARA

and

ELISA

the

capture

antibody

employed

in

a

standard

ELISA

is

static

and

solutions

are

changed

in

the

well-plate

in

a

fixed

sequence

In

FARA

the

capture

antibody

is

attached

to

a

mobile

substrate

and

solutions

are

changed

by

positioning

the

filament

in

different

solution

chambers

As

we

have

shown

previously

this

gives

FARA

a

sensitivity

limit

similar

to

ELISA

on

the

order

of

virus

particles

A

major

advantage

of

FARA

is

that

it

is

dynamic

and

allows

modification

of

processing

in

response

to

results

Virus

incubation

time

with

the

capture

antibody

can

be

reduced

to

as

low

as

one

minute

depending

on

the

virus

concentration

and

antibody

affinity

reducing

the

overall

assay

duration

On

the

other

hand

the

incubation

time

can

be

increased

in

order

to

increase

sensitivity

Moreover

because

the

filament

is

mobile

the

capture

antibody

is

brought

to

the

virus

solution

thus

enabling

use

of

capture

antibodies

with

different

specificities

to

interrogate

a

single

aliquot

of

virus

solution

Attachment

of

the

capture

antibody

to

a

mobile

filament

in

FARA

makes

accurate

positioning

of

the

filament

and

correct

identification

of

the

capture

antibody

regions

essential

for

virus

strain

classification

These

parameters

are

particularly

important

in

multilevel

testing

of

the

type

used

in

our

study

A

simple

bar

code

system

based

on

fluorescent

fiducial

markers

was

used

to

identify

the

leading

edge

of

each

of

the

capture

antibody

regions

and

position

the

filament

in

the

reaction

chambers

The

pattern

of

the

fluorescent

marks

also

was

used

to

identify

the

level

of

testing

Since

our

simple

test

system

involved

three

levels

and

testing

always

began

in

level

only

searches

for

patterns

containing

one

or

two

fluorescent

marks

corresponding

to

levels

two

and

three

were

required

However

this

simple

approach

allows

for

the

incorporation

of

more

complex

bar

codes

using

additional

spots

or

patterns

of

spots

This

study

demonstrates

the

feasibility

of

an

automated

system

for

diagnosing

specific

virus

strains

Although

the

scheme

we

report

is

a

relatively

simple

implementation

of

this

system

more

complex

designs

are

possible

For

example

each

antibody

capture

region

could

contain

additional

antibodies

In

addition

there

is

essentially

no

limit

to

the

overall

length

of

the

filament

which

would

allow

the

incorporation

of

many

additional

testing

regions

The

sensitivity

of

FARA

could

also

be

increased

by

the

use

of

quantum-dot

labeled

detection

antibodies

as

well

as

a

more

sensitive

fluorescence

detection

system

The

most

important

requirement

is

the

availability

of

a

wide

range

of

antibodies

to

the

specific

pathogens

of

interest

Such

a

strategy

may

have

applications

for

detecting

specific

pathogens

from

complex

mixtures

This

approach

would

have

both

clinical

and

environmental

applications

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

virus detection
fluorescence detection
pathogen detection


A

low

perfusion

rate

microreactor

for

continuous

monitoring

of

enzyme

characteristics

application

to

glucose

oxidase

This

report

describes

a

versatile

and

robust

microreactor

for

bioactive

proteins

physically

immobilized

on

a

polyether

sulfone

filter

The

potential

of

the

reactor

is

illustrated

with

glucose

oxidase

immobilized

on

a

filter

with

a

cut-off

value

of

kDa

A

flow-injection

system

was

used

to

deliver

the

reactants

and

the

device

was

linked

on-line

to

an

electrochemical

detector

The

microreactor

was

used

for

on-line

preparation

of

apoglucose

oxidase

in

strong

acid

and

its

subsequent

reactivation

with

flavin

adenine

dinucleotide

In

addition

we

describe

a

miniaturized

version

of

the

microreactor

used

to

assess

several

characteristics

of

femtomole

to

attomole

amounts

of

glucose

oxidase

A

low

negative

potential

over

the

electrodes

was

used

when

ferrocene

was

the

mediator

in

combination

with

horseradish

peroxidase

ensuring

the

absence

of

oxidation

of

electro-active

compounds

in

biological

fluids

A

low

backpressure

at

very

low

flow

rates

is

an

advantage

which

increases

the

sensitivity

A

variety

of

further

applications

of

the

microreactor

are

suggested

Introduction

Conventionally

characterizing

enzyme

properties

in

terms

of

kinetics

conversion

rate

and

substrate

specificity

requires

substantial

amounts

of

pure

enzyme

We

aim

to

develop

a

versatile

and

robust

microreactor

to

monitor

on-line

the

activity

of

small

amounts

of

enzymes

and

that

can

be

used

under

harsh

conditions

if

required

for

instance

to

prepare

modified

forms

of

glucose

oxidase

-LRB-

GOx

-RRB-

in

which

the

flavin

adenine

dinucleotide

-LRB-

FAD

-RRB-

cofactor

has

been

replaced

by

artificial

flavins

GOx

uses

β-d

-LRB-

-RRB-

glucose

with

high

specificity

as

substrate

converting

it

to

gluconolactone

and

Preparation

of

apoGOx

with

low

residual

activity

requires

partial

unfolding

of

the

protein

under

strongly

acidic

conditions

followed

by

removal

of

the

flavin

by

size-exclusion

chromatography

-LSB-

-RSB-

The

activity

of

the

enzyme

can

be

restored

by

applying

a

solution

of

FAD

or

FAD

analogues

-LSB-

-RSB-

Enzyme

reactors

can

be

created

by

covalent

immobilization

-LSB-

-RSB-

Another

option

is

to

co-polymerise

the

enzyme

in

a

flow

system

trapping

the

enzyme

in

a

polymer

matrix

-LSB-

-RSB-

With

these

approaches

assessment

of

enzyme

properties

inhibitor

screening

-LSB-

-RSB-

or

conversion

rates

of

stereoselective

or

enantiomeric

substrates

-LSB-

-RSB-

have

been

reported

Although

convenient

covalent

attachment

and

co-polymerisation

require

substantial

amounts

of

protein

to

obtain

a

good

response

-LSB-

-RSB-

Upon

immobilization

enzyme

characteristics

are

often

altered

and

enzyme

activity

may

be

lost

Especially

when

rare

or

expensive

enzymes

have

to

be

used

it

would

be

beneficial

to

have

a

system

that

uses

far

less

enzyme

does

not

use

chemical

immobilization

and

is

easy

to

handle

We

propose

a

device

where

the

enzyme

is

confined

in

a

small

space

on

top

of

one

filter

or

between

two

filters

and

continuously

perfused

The

device

based

on

our

previously

described

biosensor

technique

-LSB-

-RSB-

is

robust

easy

to

handle

and

loss

of

activity

and

consumption

of

enzyme

are

kept

to

a

minimum

With

this

device

we

were

able

to

prepare

apoGOx

on-line

by

retaining

GOx

on

a

single

polyether

sulfone

-LRB-

PES

-RRB-

filter

using

acid

treatment

Restoration

of

the

enzyme

activity

was

achieved

in

the

same

system

using

a

solution

of

FAD

Furthermore

we

were

able

to

miniaturize

the

microreactor

using

less

enzyme

and

increase

the

sensitivity

of

the

system

using

a

combination

of

ferrocene

and

horseradish

peroxidase

-LRB-

HRP

-RRB-

as

a

mediator

for

electrochemical

detection

Experimental

Materials

GOx

from

Aspergillus

niger

-LRB-

EC

grade

-RRB-

and

horseradish

peroxidase

-LRB-

HRP

EC

grade

-RRB-

were

obtained

from

Roche

-LRB-

Almere

The

Netherlands

-RRB-

Ferrocene

carbolic

acid

was

from

Lancaster

Chemicals

-LRB-

Lancaster

Synthetics

UK

Morecambe

Lancs

UK

-RRB-

Other

chemicals

were

of

pro

analysis

quality

and

purchased

from

Merck

-LRB-

Amsterdam

The

Netherlands

-RRB-

Double

quartz-distilled

water

was

used

for

all

aqueous

solutions

Before

and

after

acid

treatment

the

running

buffer

-LRB-

a

-RRB-

of

the

microreactor

was

mol

L

sodium

phosphate

pH

containing

mmol

L

NaCl

and

Kathon

GC

-LRB-

Rohm

and

Haas

Croydon

Surrey

UK

-RRB-

Glucose

solution

-LRB-

mmol

L

-RRB-

prepared

in

running

buffer

was

left

to

stand

for

several

hours

to

reach

mutarotational

equilibrium

For

electrochemical

detection

of

GOx

in

combination

with

HRP

the

running

buffer

-LRB-

b

-RRB-

also

contained

mmol

L

ferrocene

carbolic

acid

Design

of

the

microreactors

The

overall

design

of

the

microreactors

is

shown

in

Fig.

They

were

micro

machined

from

Delrin

by

the

instrumental

workshop

of

Groningen

University

For

preparation

of

apoGOx

and

reconstitution

of

GOx

activity

a

microreactor

as

described

in

Ref

-LSB-

-RSB-

was

used

In

contrast

to

the

original

device

only

one

PES

filter

with

a

cut-off

value

of

kDa

-LRB-

Sartorius

Göttingen

Germany

-RRB-

and

punched

to

a

diameter

of

mm

was

used

Performance

studies

were

done

with

our

miniaturized

design

The

dimensions

of

the

miniaturized

microreactor

were

outer

length

cm

outer

diameter

cm

A

PES

membrane

filter

was

punched

to

a

diameter

of

mm

and

placed

in

the

microreactor

-LRB-

effective

diameter

mm

effective

volume

μL

-RRB-

and

connected

between

the

injection

valve

and

the

detector

with

fused-silica

tubing

-LRB-

FST

-RRB-

-LRB-

μm

OD

μm

ID

-RRB-

-LRB-

Polymicro

Technologies

Phoenix

AZ

USA

-RRB-

pinched

off

in

Teflon

tubing

inch

OD

and

μm

ID

-LRB-

Aurora

Borealis

Control

Schoonebeek

The

Netherlands

-RRB-

with

use

of

finger-tight

fittings

to

avoid

high

dead

volumes

Fig.

design

of

the

reaction

compartment

of

the

microreactor

-LRB-

a

-RRB-

sketch

-LRB-

b

-RRB-

schematic

diagram

Shown

here

is

the

microreactor

with

one

PES

ultrafilter

which

is

then

connected

to

the

flow

system

using

FST

and

finger-tight

fittings

The

mesh

screen

is

used

to

support

the

ultrafilter

Set-up

of

the

system

The

perfusates

of

the

microreactor

were

introduced

into

a

flow-injection

system

and

monitored

electrochemically

-LRB-

electrochemical

flow

cell

and

Decade

Digital

Electrochemical

Amperometric

Detector

Antec

Leyden

Zoeterwoude

The

Netherlands

-RRB-

essentially

as

previously

described

-LSB-

-RSB-

For

the

preparation

of

apoGOx

and

subsequent

reconstitution

of

the

holoenzyme

a

voltage

of

V

was

applied

between

the

working

and

reference

electrodes

GOx/HRP

activity

monitoring

For

flows

μL

min

a

solvent-delivery

pump

-LRB-

Shimadzu

Corporation

Kyoto

Japan

-RRB-

was

used

for

flows

μL

min

a

Harvard

pump

-LRB-

Harvard

Apparatus

Holliston

MA

USA

-RRB-

was

used

A

voltage

of

mV

was

applied

between

the

working

-LRB-

glassy

carbon

-RRB-

and

reference

-LRB-

Ag/AgCl

-RRB-

electrodes

The

injection

valve

-LRB-

Vici

Cheminert

Valco

Instruments

Houston

TX

USA

-RRB-

was

equipped

with

a

nL

internal

loop

injection

cycles

were

designed

using

software

incorporated

in

the

Decade

system

which

also

thermostatically

controlled

the

reaction

temperature

at

°C

The

current

was

registered

using

a

flatbed

recorder

type

-LRB-

Kipp

Zonen

Delft

The

Netherlands

-RRB-

and/or

the

signal

was

integrated

using

Chromeleon

Software

-LRB-

Dionex

Corporation

Sunnyvale

CA

USA

-RRB-

on

a

PC

using

an

interface

After

the

filter

was

installed

in

the

system

it

was

left

to

equilibrate

to

a

flow

of

nL

min

Glucose

solution

injection

was

sequenced

to

load/inject

min

-LRB-

cycle

time

min

-RRB-

and

repeated

until

a

stable

baseline

was

obtained

Preparation

of

apoGOx

and

restoration

of

the

enzyme

activity

Preparation

of

apoGOx

was

essentially

the

same

as

described

in

Ref

-LSB-

-RSB-

with

a

solution

of

mol

L

glycine

mol

L

NaCl

and

mol

L

HCl

pH

mixed

with

glycerol

to

a

final

concentration

of

glycerol

of

-LRB-

v/v

-RRB-

cooled

in

ice

This

solution

was

applied

to

the

GOx

containing

reactor

In

detail

a

μL

solution

of

mg

mL

GOx

-LRB-

μmol

L

-RRB-

was

pipetted

on

to

the

microreactor

's

PES

filter

which

was

thus

loaded

with

nmol

After

stabilisation

of

the

signal

the

initial

enzyme

activity

was

determined

-LRB-

-RRB-

The

reactor

was

detached

from

the

detector

The

detached

reactor

was

cooled

in

ice

and

perfused

with

the

cooled

acidic

glycerol

solution

at

a

rate

of

μL

min

for

min

After

application

of

running

buffer

-LRB-

a

-RRB-

until

the

effluent

was

pH

the

reactor

was

taken

out

of

the

flow

system

re-attached

to

the

detector

cell

and

the

residual

activity

of

the

apoGOx

was

measured

in

buffer

system

-LRB-

a

-RRB-

with

a

flow

of

μL

min

After

the

reactor

was

released

from

the

flow

system

ca

μL

of

an

FAD

solution

-LRB-

mmol

L

-RRB-

in

mol

L

sodium

phosphate

pH

at

room

temperature

was

applied

manually

during

ca

min

except

for

time-dependent

tests

Afterwards

the

reactor

was

re-attached

to

the

detector

cell

and

the

restored

activity

of

the

holoenzyme

was

measured

as

described

above

Determination

of

several

characteristics

of

the

miniaturized

enzyme

reactor

Increasing

amounts

of

GOx

in

running

buffer

-LRB-

b

-RRB-

-LRB-

range

amol

to

pmol

-RRB-

were

injected

into

the

system

providing

the

amount

of

GOx

retained

on

the

membrane

After

equilibration

a

sequence

of

load/inject

of

the

glucose

solution

was

applied

Subsequently

a

solution

of

HRP

in

running

buffer

-LRB-

b

-RRB-

was

injected

into

the

system

providing

pmol

-LRB-

ng

-RRB-

of

HRP

After

equilibration

the

sequence

of

load/inject

of

glucose

was

repeated

The

efficiency

of

conversion

at

the

electrode

was

calculated

according

to

Eq

-LRB-

-RRB-

where

h

peak

height

-LRB-

pA

-RRB-

σ

peak

width

at

-LRB-

s

-RRB-

I

injection

volume

-LRB-

μL

-RRB-

c

concentration

-LRB-

μmol

L

-RRB-

n

number

of

electrons

F

-LRB-

Faraday

's

constant

C

mol

-RRB-

and

-LRB-

2π

-RRB-

hσ

is

peak

area

-LRB-

C

-RRB-

In

Eq

-LRB-

-RRB-

the

charge

that

is

transferred

to

the

electrode

-LRB-

area

under

the

curve

-RRB-

is

related

to

the

real

charge

that

is

available

in

the

reaction

e.g.

the

concentration

of

the

substrate

the

number

of

electrons

involved

and

Faraday

's

constant

Results

Preparation

of

apoGOx

and

restoration

of

the

enzyme

activity

We

followed

the

response

to

glucose

the

residual

enzyme

activity

of

apoGOx

after

acid

treatment

and

restoration

of

the

enzyme

activity

after

application

of

a

solution

of

FAD

After

acid

treatment

negligible

activity

of

apoGOx

was

observed

Application

of

the

FAD

solution

restored

the

enzyme

activity

The

time

dependency

of

enzyme

activity

restoration

is

shown

in

Fig.

composed

of

individual

data

from

five

apoGOx

preparations

This

time

dependency

of

the

reactivation

is

in

accordance

with

Eq

-LRB-

-RRB-

where

k

min

and

t

is

the

reaction

time

-LRB-

min

-RRB-

Fig.

of

apoGOx

activity

circles

observed

relative

reactivation

with

FAD

solid

line

fitting

according

to

the

equation

f

-LRB-

t

-RRB-

exp

-LRB-

t

-RRB-

Approximately

of

the

enzyme

activity

was

restored

within

min

which

is

above

average

compared

to

other

procedures

-LSB-

-RSB-

Performance

Several

properties

of

the

system

were

investigated

These

included

backpressure

of

the

reactor

at

various

protein

loads

its

stability

and

reproducibility

the

efficiency

at

the

electrode

system

sensitivity

and

ease

of

handling

The

relationship

of

backpressure

to

the

flow

and

the

amount

of

protein

retained

on

the

filter

are

depicted

in

Figs.

and

The

level

of

the

backpressure

is

acceptable

with

these

flows

and

these

amounts

of

protein

The

stability

and

reproducibility

of

the

system

as

tested

with

fmol

GOx

on

a

series

of

glucose

injections

showed

an

intra-assay

standard

deviation

of

less

than

The

efficiency

of

the

reaction

is

inversely

proportional

to

the

amount

of

protein

on

the

filter

The

sensitivity

of

the

set-up

was

determined

by

retaining

various

amounts

of

GOx

ranging

from

amol

to

pmol

on

the

ultrafilter

and

testing

the

response

A

measurable

and

reproducible

response

was

obtained

even

with

the

lowest

amounts

of

enzyme

tested

-LRB-

Table

-RRB-

One

load

of

enzyme

in

the

microreactor

can

be

used

at

least

times

and

the

system

can

be

regenerated

simply

by

replacing

the

filter

and

injection

of

a

fresh

aliquot

of

protein

solution

Fig.

backpressure

-LRB-

a

-RRB-

Backpressure

in

relation

to

the

flow

at

various

protein

loadings

diamonds

μg

squares

μg

triangles

μg

circles

μg

stars

μg

GOx

-LRB-

b

-RRB-

Backpressure

in

relation

to

the

amount

of

protein

retained

on

the

filter

at

various

flow

rates

circles

μL

min

squares

μL

min

triangles

μL

min

diamonds

μL

min

detector

signal

and

relative

conversion

of

glucose

in

relation

to

the

amount

of

enzyme

retained

on

the

filter

in

the

miniaturized

microreactor

Indicated

are

the

amounts

of

GOx

-LRB-

in

fmol

-RRB-

on

the

PES

filter

and

the

signals

of

the

detector

-LRB-

in

-RRB-

indicating

the

amount

of

converted

glucose

calculated

as

described

in

the

method

sectionProtein

-LRB-

GOx

-RRB-

on

the

filter

-LRB-

fmol

-RRB-

Signal

-LRB-

-RRB-

Conversion

-LRB-

-RRB-

0.06250.0160.040.6250.0280.0066.250.0510.0016250.530.00016250002.40.0000006

Conclusions

The

low

perfusion

rate

microreactor

is

suitable

for

the

new

application

described

here

preparation

of

minute

amounts

of

apoGOx

and

reconstitution

of

enzyme

activity

with

a

solution

of

FAD

In

comparison

to

the

conventional

preparation

of

apoGOx

using

size-exclusion

chromatography

-LSB-

-RSB-

this

device

requires

neither

complicated

handling

nor

expensive

materials

and

equipment

The

enzyme

is

simply

retained

on

the

PES

ultrafilter

A

scale-up

of

the

microreactor

can

easily

be

performed

allowing

the

preparation

of

larger

amounts

of

apoGOx

to

be

used

for

example

in

studies

with

modified

FAD

-LSB-

-RSB-

or

as

the

basis

of

a

biosensor

with

which

a

wide

variety

of

analytes

can

be

detected

-LSB-

-RSB-

The

combination

with

immunological

principles

allows

the

analysis

of

small

molecules

when

no

specific

enzyme

is

available

combining

molecular

recognition

with

enzyme

enhancement

of

the

signal

The

sensitivity

is

improved

even

more

by

using

electrochemical

detection

Low

perfusion

rate

microreactors

may

facilitate

the

study

of

enzymatic

properties

and

modifications

including

substrate

inhibition

competitive

and

non-competitive

inhibition

and

allosteric

activation

Because

of

the

mild

fixation

conditions

they

may

enable

investigation

of

receptor

activity

also

An

advantage

of

the

microreactor

presented

here

is

the

small

amount

of

protein

required

thus

allowing

the

use

of

expensive

and

rare

enzymes

Applications

using

related

devices

e.g.

Refs

-LSB-

-RSB-

usually

require

micrograms

to

milligrams

of

protein

Glucose

sensors

using

glucose

oxidase

or

glucose

dehydrogenase

have

also

been

developed

for

clinical

use

One

example

of

such

an

application

uses

a

volume

of

nL

blood

for

glucose

measurements

in

diabetics

-LSB-

-RSB-

Such

reactors

still

use

larger

volumes

as

compared

to

the

nL

we

used

here

Although

we

used

electrochemical

detection

other

modes

of

detection

including

capillary

electrophoresis

fluorescence

or

chemiluminescence

are

possible

In

addition

with

the

current

interest

in

proteomics

applications

can

be

found

where

minute

amounts

of

protein

can

be

digested

reproducibly

and

analysed

directly

by

LC

MS

MS

-LSB-

-RSB-

O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

apoglucose oxidase
enzyme reactor
flavin adenine dinucleotide
glucose oxidase