ANALYTICAL

BIOCHEMISTRY
Analytical Biochemistry 334 (2004) 196198
www.elsevier.com/locate/yabio

Notes & Tips

A spectrophotometric assay for the quantiWcation

of polyethylenimine in DNA nanoparticles
Martin Bertschinger, Sophie Chaboche, Martin Jordan, Florian M. Wurm
Swiss Federal Institute of Technology Lausanne, Laboratory of Cellular Biotechnology, Lausanne VD1015, Switzerland
Received 29 April 2004
Available online 28 August 2004

Since the Wrst description of polyethylenimine (PEI)1 as

a gene delivery vehicle, there has been a strong interest in
its use for in vivo and in vitro transfections of mammalian
cells [1]. The mechanism of gene transfer by PEI is still
unclear, and little is known about the formation of PEI
DNA nanoparticles. Such questions are diYcult to address
because a rapid and sensitive assay for the quantiWcation
of PEI in diVerent transfection media is not available.
The only spectrophotometric assay for the quantiWcation of PEI is based on the dark blue cuprammonium
complex that is formed when copper(II) ions are added to
PEI [2,3]. This complex has a strong absorbance peak at
285 nm and a weaker one at 630 nm. Unfortunately, the
absorption maximum at 285 nm is close to that of DNA
(260 nm). In addition, most cell culture media contain
components that absorb at this wavelength. At 630 nm,
there is much less interference from biological molecules,
but the signal from the cuprammonium complex is also
low. We successfully adapted a commercially available
protein detection assay for the quantiWcation of PEI.
All experiments described below were performed with
linear 25-kDa PEI (Polysciences, Warrington, PA, USA)
that we routinely use for the transfection of mammalian
cells [4], but it was possible to quantitate other PEIs
(diVerent molecular weights; branched) with this method
(data not shown). The mixing of a PEI solution with
the Advanced Protein Assay (APA) reagent (Cytoskeleton, Denver, CO, USA) resulted in the formation of a
colored product that absorbed in the range of 570
615 nm, with the absorbance being proportional to the
*

Corresponding author.
E-mail address: martin.jordan@epX.ch (M. Jordan).
1
Abbreviations used: PEI, polyethylenimine; QOD, limit of quantiWcation; LOD, limit of detection.
0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2004.07.020

PEI concentration. BrieXy, 200
l of the APA reagent

was mixed with 800
l of the PEI sample by vortexing
for 5 s. The solution was then incubated for 30 min at
room temperature before the absorbance was measured
at 590 nm. For incubation periods of less than 30 min, the
absorbance signal was more variable than was observed
for a 30-min incubation. Incubation periods of more
than 30 min frequently resulted in visible precipitates.
We initially quantiWed PEI in 150 mM NaCl because
DNA and PEI are frequently mixed in this salt solution
prior to transfection. The limit of quantiWcation (QOD,
estimated to be 10 times the standard deviation of
blanked values) was approximately 50 ng/ml or 100
times lower than had been determined previously [3].
The limit of detection (LOD, estimated to be three times
the standard deviation of blanked values) was 16 ng/ml.
The absorption increased linearly up to a PEI concentration of 1
g/ml (Table 1). Using a nonlinear four-parameter curve Wt (Softmax Pro 2.4, Molecular Devices,
Sunnyvale, CA, USA), PEI concentrations of up to 4
g/
ml were measured without dilution (Fig. 1).
PEI standard curves were also established in diVerent
cell culture media. The absorbance maximum of the
APA reagent is suitable for these measurements. Because
the only component in media that absorbs at 590 nm is
phenol red, the low pH of the APA solution resulted in a
complete protonation of phenol red, eliminating its
interference at this wavelength. In RPMI 1640 (Applichem, Darmstadt, Germany), the PEI concentration was
measured with nearly the same sensitivity as in 150 mM
NaCl (QOD D 0.2
g/ml). However, chemically deWned
media such as EX CELL 293 (JRH Bioscience, Lenexa,
KS, USA) and ProCHO5 (Cambrex, East Rutherford,
NJ, USA) reacted with the APA reagent. To reduce the
background due to unknown medium components, the

Notes & Tips / Analytical Biochemistry 334 (2004) 196198

197

Table 1
Optical characteristics and limitations of the PEI assay at  D 590 nm
Medium

Dilution
LOD (
g/ml)
QOD (
g/ml)
Linear range (
g/ml)

NaCl (150 mM)

EXCELL 293

RPMI 1640

ProCHO5

0.016
0.053
0.051

1:25
1.13
3.8
3.850

0.059
0.2
0.22.0

1:25
0.57
1.9
2.050

Fig. 1. Standard curve of PEI concentration in 150 mM NaCl. The

absorbances of solutions with known concentrations of PEI were
determined at 590 nm following incubation with the APA reagent. The
absorbance values were blanked against air and Wtted using a nonlinear four-parameter curve as described in the given equation. The linear
range of the standard curve is shown in magniWcation.

samples were Wrst diluted 25-fold with 150 mM NaCl.

Unfortunately, this decreased the QOD for PEI to 4
g/
ml in EX CELL 293 and to 2
g/ml in ProCHO5 (Table
1). The linear ranges for the three diVerent media are
summarized in Table 1.
We also analyzed the inXuence of DNA on PEI quantiWcation using the APA reagent and found that the
reagent has a more than 150-fold higher aYnity for PEI
than for DNA (OD/
g of DNA D 0.002 vs. OD/
g of
PEI D 0.36). Therefore the eVect of DNA on the measurement was neglected for the experiment described
below.
When plasmid DNA and PEI are mixed, they immediately form a complex [5]. At higher PEI concentrations,
these complexes are further compacted into PEIDNA
nanoparticles that can be sedimented in a microcentrifuge at 12,800g for 2 min. Nanoparticle formation was
observed when the PEI:DNA ratio (w/w) in the mixture
was greater than 0.25 (Fig. 2). The complete incorporation of DNA into particles was demonstrated by spectrophotometric analysis of the supernatant after
centrifugation. All of the DNA was present in the pellet
fraction at PEI:DNA ratios of 0.25 or more (Fig. 2).
Using the APA reagent, it was also possible to quantify
the PEI in solution. Without sedimentation, both soluble

Fig. 2. QuantiWcation of PEI and DNA in diVerent mixtures after

removal of PEIDNA nanoparticles by centrifugation. Two equal volumes of 150 mM NaCl with either 50
g/ml DNA or varying amounts
of PEI were mixed, incubated for 10 min at room temperature, and
then centrifuged for 2 min at 12,800g in a microcentrifuge. Undiluted
supernatants were used to quantify the DNA concentration at 260 nm.
For the PEIAPA assay, supernatants were diluted 50-fold in 150 mM
NaCl to be in the range of the calibration curve. The dashed line represents the added amount of PEI.

PEI and PEI complexed to DNA were detected. After

removal of the nanoparticles by centrifugation, only the
PEI remaining in solution was detected (Fig. 2). At the
critical PEI:DNA ratio of 0.25, no PEI remained in solution because all of it was present in nanoparticles.
Increasing the PEI did not result in a further incorporation of PEI into nanoparticles. At PEI:DNA ratios
greater than 0.25, the PEI concentration in the supernatant increased linearly with the amount of PEI added,
demonstrating that the PEI:DNA complexes formed at
ratios of 0.25 to 2 contained the same amount of PEI.
This result was surprising given that the PEIDNA complexes formed at ratios between 0.25 and 0.50 were not
competent for transfection, but high transfection eYciencies were obtained with PEI:DNA ratios of 2 or more [4].
We showed that the APA reagent is an excellent tool
for quantiWcation of PEI in a concentration range from
0.05 to 4
g/ml in 150 mM NaCl. Moreover, the interaction between this reagent and PEI was not disturbed by
most cell culture media or by the presence of DNA. The

198

Notes & Tips / Analytical Biochemistry 334 (2004) 196198

method is fast, involves little manipulation, and exceeds

the sensitivity of previously published spectrophotometric methods. With this assay, it was demonstrated that
PEI:DNA particles form at a critical PEI:DNA ratio of
0.25 while incorporating all of the available PEI and
DNA molecules. At this ratio, the DNA was fully saturated with PEI, and further addition of soluble PEI did
not result in incorporation into the nanoparticles. With
this assay, a systematic analysis of PEI:DNA complex
formation in diVerent standard cell culture media is possible and may help to optimize transfection.

Acknowledgment
The authors thank David Hacker for critically reading the manuscript.

References
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