The study and use of bioluminescence and chemiluminescence
have increased dramatically in recent
years. Applications range from adenosine triphosphate
(ATP)–luciferase assay and cell viability tests
to immunoassay, DNA, genomic, and proteomic
analyses. Unlike fluorescence systems, there is no
need for an exogenous light source, since luminescence
is generated from chemical reactions.
Utilizing photon-counting detectors, which count the
number of photons generated from a chemical reaction,
the luminescence assay has become one of the most
sensitive and quantitative optical detection methods.
During the past 15 years, nucleic acid (NA) hybridization
techniques have become increasingly important as
discovery tools and for the clinical diagnosis of genetic
disorders and infectious diseases.1–3 These methodologies
have benefited greatly from the development of NA amplification techniques that have significantly
improved the sensitivity of detection of nucleic acids
such as DNA and RNA and their chimeric derivatives.
Nucleic acid amplification is suitable for low-abundant
gene transcripts and genotyping of rare allele mutations,
as well as for the measurement of low-copy-number
RNA and DNA pathogens (i.e., bacteria and viruses)
in diverse biological sources, particularly in applications
that depend on extremely limited nucleic acid samples,
such as cancer diagnostics, pharmacogenomics, toxicogenomics,
forensics, the food industry, and environmental
Molecular diagnostics and
Figure 1 - Magnetic bead-based DNA hybridization assay with a
label HRP for chemiluminescence detection. Once the substrate is added,
the reaction produces luminescence.
Figure 2 - The magnetic bead-based sandwich hybridization assay has
a detection limit between 1 and 5 attomole of target oligonucleotide and a
dynamic range of 3–4 orders of magnitude.
Figure 3 - The LuminMax-C offers attomole sensitivity by
quantitative photon counting.
The following section describes a sandwich hybridization
assay for quantitative oligonucleotide target detection.
The assay combines 1) a magnetic bead-based capture
probe, 2) target DNA hybridization, and 3) chemiluminescent
target detection. For the assay, 5′ end biotinylated
single-stranded capture oligonucleotides (MWG Biotech,
Highpoint, NC), representing a 30-bp sequence within
the coding region of human glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), was attached to streptavidin-coated
paramagnetic beads (Dynal MyOne™, Invitrogen
Corp., Carlsbad, CA) via the biotin–streptavidin
bonding. Magnetic beads (20 μg) were incubated with
20 pg of capture oligonucleotide (5′ biotin-GCT CTT
TGC ACT GGT AGA CAG AGA TCT CAT-
3′) in 10 mM Tris–HCl (pH 7.5)/1 mM ethylenediaminetetraacetic
acid (EDTA)/2 M NaCl for 20 min
at room temperature. Unbound material was washed
off, and the beads were resuspended in 20 μL of the
same buffer (this can be stored at 4 °C for several
months). The assembled capture oligo-beads were
washed and resuspended in phosphate-buffered saline
(PBS) prior to hybridization with a 30-mer target oligonucleotide
with the exact complementary sequence
plus carrying a digoxigenin molecule at its 5′ end
(5′digoxigenin-ATG AGA TCT CTG TCT ACC
ABT GCA AAG AGC -3′) (Figure 1). Hybridization
was performed in hybridization buffer (MWG
Biotech) for 90 min at 42 °C using a thermal cycler. The hybridized product was subsequently
blocked with 2% bovine serum albumin (BSA)/0.1%
Tween 20/PBS for 30 min at room temperature before
adding 3 mU of a sheep antidigoxigenin polyclonal
antibody conjugated to horseradish peroxidase (HRP)
(Roche Applied Sciences, Indianapolis, IN) to a final
concentration of 0.15 mU/μL. The immunodetected
beads were washed four times with 1 mL of 1% Tween
20/PBS and finally with PBS before pelleting and
resuspending in 20 μL of PBS for transfer to a black
clear-bottom microplate (cat. no. 265301, Nalge
Nunc International Corp., Rochester, NY). Chemiluminescent
signal enhancement was achieved by
adding HRP LuminMax Super™ substrate (Maxwell
Sensors, Santa Fe Springs, CA) and recorded (Figure
2) with a LuminMax-C™reader (Figure 3).
Figure 4 - The dynamic range of the polyclonal antibody reaches
over seven orders of magnitude from 30 mU to 10–5 mU using the same
amount of LuminMax Super HRP substrate.
The LuminMax-C reader offers microplate setting
for cell biology, molecular biology, and immunology
applications in the fields of cancer research, drug
development, proteomics, and genomics. It is also
suitable for testing antibody activity prior to the use
of antibodies in ELISAs such as those utilized modularly
for the hybridization sandwich assay described
above. The authors tested the sensitivity of the antidigoxigenin
antibody in a titration assay using different
antibody concentrations, while keeping the
amount of chemiluminescent substrate constant
(Figure 4). The compact, economical system offers
high sensitivity, accuracy, and ease of use. It measures
the luminescence intensity in a 96-well microplate
(black or white). Because the microplate has a clear
bottom, luminescence can be accurately detected
from the bottom of the well. Since it uses a state-of-the-art photon-counting multiplier tube as the
detector, the system is able to count a single photon generated from the reaction, and thus it can detect very
small amounts of analyte in the samples. A CD, with
user-friendly software, is provided for easy installation.
The system utilizes a PC or notebook as its microprocessor.
It is interfaced to a computer by a simple plug-in
(USB or serial port) connection. After clicking the “Go”
button, the system automatically and quickly scans all of
the selected microplate wells and shows the results. The
resulting data are displayed as a spreadsheet in Microsoft®
Excel™ (Microsoft, Redmond, WA) format.
- Qureshi, M.N.; Bolick, D.; Ringer, P.J.; Spagler, F.L.;
Zimmerman, G. HPV testing in liquid cytology specimens:
comparison of analytic sensitivity and specificity
for in situ hybridization and chemiluminescent
nucleic acid testing. Acta Cytol. 2005, 49(2), 120–6.
- Leal, E.; Jaloma-Cruz, A.R.; Barros-Nunez, P. High
sensitivity of chemiluminescent methodology for
detection of clonal CDR3 sequences in patients with
acute lymphoblastic leukemia. Hematol. Oncol. 2004,
- Patel, R.; Pollner, R.; de Keczer, S.; Pease, J.; Pirio, M.;
DeChene, N.; Dafforn, A.; Rose, S. Quantification of
DNA using the luminescent oxygen channeling assay.
Clin. Chem. 2000, 46(9), 1471–7.
Mr. Ho is a Research Intern, and Dr. Tajbakhsh is a Senior
Research Scientist, Maxwell Sensors Inc., 10020 Pioneer Blvd.,
Ste. 103, Santa Fe Springs, CA 90670, U.S.A.; tel.: 562-801-2088; fax: 562-801-2089; e-mail: firstname.lastname@example.org.